1 CHLAMYDIA TRACHOMATIS CAUSES CHROMOSOME SEGREGATION ERRORS LEADING TO CYTOKINESIS FAILURE By HEATHER MARIE BROWN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQ UIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Heather Marie Brown
3 To G randdad, for always being one of my biggest fans
4 ACKNOWLEDGMENTS I would like to thank my parents for their love and unending support. I would have not made it through my PhD without my mom coming to keep me company and cook me delicious meals when I had an awful week and my dad reminding me to take it one day at a time. I would like to thank my brothers, Erik and Alex, for being protective and my partner in crime. I would like to thank my mentor Dr. Scott Grieshaber for your amazing patience. I know it took a lot of work to get me from only being able to take pictures on the microscope to being able to change a mercury bulb and trouble shoot all the crazy things people do to our scope. I would also like to thank my committee members Dr. Edward Chan, Dr. Paul Gulig, Dr. Brian Law, and Dr. Daniel Purich. Thank you for taking your time out of your schedule to meet with me and give helpful advice. I was sad when I was told my third aim was boring, but in the end it really strengthened my project. Next, I would like to thank Dr. Nicole Grieshaber and Dr. Shannon Wallet for all their great advice and help. Its wonderful to have auxiliary mentors for extra support. Finally I would like to thank the lab, I wouldnt have made it through the program, without having a group to commit shenanigans with. Specifically, without Andrea I might have quit and become a zookeeper. Instead, Im now an expert on Chlamydia.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Chlamydia ............................................................................................................... 13 Host Cell ................................................................................................................. 16 Effects on the Cell Cycle .................................................................................. 16 Effects on the Centrosome Cycle ..................................................................... 20 Cancer Biology ....................................................................................................... 21 2 MATERIALS AND METHODS ................................................................................ 26 Organisms and Cell Culture .................................................................................... 26 Infections ................................................................................................................ 26 Immunohistochemistry ............................................................................................ 26 Transfections and Plasmids .................................................................................... 27 Live Cell Imaging .................................................................................................... 28 Cell Fusion Assay ................................................................................................... 28 Inclusion Measurement ........................................................................................... 28 CellFree Degradation Assay .................................................................................. 28 Mitotic Index Assays ............................................................................................... 29 Western Blot ........................................................................................................... 29 Double Thymidine Block ......................................................................................... 30 Fluorescence Ubiquinitation Cell Cycle Indicator .................................................... 30 Griseofulvin Treatment ............................................................................................ 30 Centrosome Calculations ........................................................................................ 31 Statistical Analysis .................................................................................................. 31 3 CHLAMYDIA INFECTION INDUCES HOST CYTOKINESIS FAILURE AT ABCISSION ............................................................................................................ 33 Introduction ............................................................................................................. 33 Results .................................................................................................................... 35 Cytokinesis Fails Very Late in Infected Cells .................................................... 35
6 Multin ucleation is Not Caused by Fusion .......................................................... 36 Cytokinesis Fails After Ingression .................................................................... 37 Infected Dividing Cells Make Midbodies ........................................................... 39 Lagging Chromosomes are Increased in Frequency in the Midbody of Infected Cells and Correlate with Multinucleation .......................................... 40 Chlamydial Infected C ells do not Arrest in Metaphase ..................................... 41 Discussion .............................................................................................................. 45 4 DECLUSTERING OF HOST CELL CENTROSOMES IS NOT CONSERVED AMONG SPECIES OF CHLAM YDIA ...................................................................... 58 Introduction ............................................................................................................. 58 Results .................................................................................................................... 60 Chlamydia Species Cause Different Rates of Multinucleation .......................... 60 Treating Cells with Griseofulvin Increases Multinucleation Rate ...................... 61 Centrosomes Are Mislocalized and Declustered during Interphase ................. 62 Declustered Centrosomes Cause a Disorganized Mitosis ................................ 63 Discussion .............................................................................................................. 65 5 DISCUSSION ......................................................................................................... 76 LIST OF REFERENCES ............................................................................................... 82 BIOGRAPHICAL SKETCH ............................................................................................ 92
7 LIST OF TABLES Table page 2 1 Inhibitors used in experiments. ........................................................................... 32 2 2 Peptide Inhibitors ................................................................................................ 32
8 LIST OF FIGURES Figure page 1 1 The cell cycle.. .................................................................................................... 24 1 2 Centrosome Cycle.. ............................................................................................ 25 3 1 Multinucleation of host cells is due to cytokinesis failure and not cell fusion. ..... 49 3 2 Chlamydia does not sterically hinder cytokinesis.. .............................................. 50 3 3 Chlamydia infected cells form midbodies .......................................................... 51 3 4 Chlamydia induces lagging chromosomes in infected cells ................................ 52 3 5 Chlamydia infection causes the premature exit of mitosis. ................................. 54 3 6 CPAF degrades cyclin B1 and securin. .............................................................. 55 3 7 Host cell entry into mitosis .................................................................................. 56 3 8 Model of chlamydial induced multinucleation. ..................................................... 57 4 1 Multinucleation with different species ................................................................. 68 4 2 Declustering of centrosomes by griseofulvin ...................................................... 70 4 3 Centrosome to nucleus distance. ....................................................................... 71 4 4 Centrosome clustering in ne uroblastoma cells .................................................. 72 4 5 Mitosis is disorgani zed upon infection with L2 .................................................... 73 4 6 DNA segregation errors correlate with multinucleation ....................................... 74 4 7 Model of differences in multinucleation. ............................................................. 75
9 LIST OF ABBREVIATIONS APC anaphase promoting complex CCV Coxiella containing vacuole CDK1 cyclin dependent kinase1 CPAF chlamydial proteaselike activity factor DNA deoxyribonucleic acid EB ele mentary body FISH Fluorescence in situ hydrization FUCCI fluorescence ubiquitination cell cycle indicator G Chlamydia trachomatis serovar G G1 gap phase G2 second gap phase GAPDH glyceraldehyde 3phosphate dehydrogenase GFP green fluorescent protein GPIC Chlamydia caviae (Guinea Pig Infective Conjunctivitis) HBSS Hanks Balanced Salt Solution HPV human papillomavirus IRA International Research Agency IFU inclusion forming unit KDA kilodalton L2 Chlamydia trachomatis serovar L2 M molar MOI multiplicity of infection MOPN Chlamydia muridarum (Mouse pneumonitis)
10 MTOC microtubule organizing center NMBD nuclear membrane breakdown NUMA nuclear mitotic apparatus protein PBS phosphate buffered saline PCM pericentriolar m aterial PCR polymerase chain reaction RB reticulate body RFP red fluorescent protein S synthesis phase SAC spindle assembly checkpoint SEM standard error of the mean STI sexually transmitted infection TARP translocated actinrecruiting phosphoprotein UV ultraviolet
11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHLAMYDIA TRACHOMATIS CAUSES CHROMOS OME SEGREGATION ERRORS LEADING TO CYTOKINESIS FAILURE By Heather Marie Brown May 2013 Chair: Scott S. Grieshaber Major: Medical Sciences Immunology and Microbiology Chlamydiae are obligate intracellular bacteria that cause a variety of diseases in both humans and animals. Infection with Chlamydia trachomatis can lead to host cell multinucleation. Our goal was to determine the mechanism by which a chlamydial infection leads to cytokinesis failure. We demonstrated that cytokinesis failure is not due to steric interference and through live cell imaging showed that cytokinesis failure occurs at abscission. Cells that failed in cytokinesis contained DNA in the midbody of the dividing cell. Therefore we investigated changes in mitosis. Infected cells hav e a reduced mitotic index suggesting that infected cells spend less time in mitosis, which allows less time to organize chromosomes. Furthermore, we demonstrated that C. trachomatis infected cells are able to escape a monastrol block, suggesting a premat ure exit fr om mitosis. We conclude that C. trachomatis induce a premature exit from mitosis that causes DNA in the midbody of the cell, resulting in cytokinesis failure at abscission. Next, we investig ated if cytological changes were conserved among chlam ydial species. C. trachomatis L2 causes shortened mitosis, declustering of amplified centrosomes and cytokinesis failure resulting in multinucleation. We compared C. trachomatis to Chlamydia muridarum and Chlamydia caviae to determine the
12 conservation of these effects between different Chlamydia species. All three species were able to cause a decrease in mitotic index and amplified centrosomes. We observed a greater percentage of multinucleated cells and multipolar spindles due to C. trachomatis infect ion compared to the other Chlamydia species. The spindle multipolarity was the only cytological change that followed the observed trend in multinucleation. We found that spindle multipolarity correlated to centrosome displacement and spread during interphase in infected cells. These results suggest that Chlamydia species may interact differently with host centrosomes, resulting in varying rates of multinucleation. We hypothesize that different cytological changes among species correlate with the ability to cause transformation.
13 CHAPTER 1 INTRODUCTION Chlamydia Chlamydiae are gram negative obligate intracellular bacteria that require the entry into mucosal epithelial cells to grow and replicate (Swanson et al., 1975; Moulder, 1991). Chlamydia consist s of a large group of species that have the ability to infect organisms as diverse as amoebas and mammals (Wheelhouse and Longbottom, 2012). Different disease etiologies can occur depending on the serovar, even within the same species. The ocular serovars of the human pathogen Chlamydia trachomatis cause trachoma, the leading cause of infectious blindness worldwide (Burton and Mabey, 2009). The urogenital serovars are the causative agent of chlamydia, the most commonly reported STI worldwide with an estimated 9 2 million new cases annually (WHO, 2001). Genital infections are often subclinical and therefore left untreated, allowing the infection to become chronic. Chronic infections can lead to serious health sequelae such as pelvic inflammatory disease and tubal infertility in untreated women (Cates and Wasserheit, 1991). Chlamydia has also been clinically associated with cytologic cervical atypia (Kiviat et al., 1985) and epidemiologically linked to an increase in the risk for cervical cancer (Anttila et al., 2001; Smith et al., 2002; Madeleine et al., 2007). A direct molecular mechanism responsible for this link has yet to be described. Chlamydiae undergo a unique biphasic developmental cycle. During the developmental cycle Chlamydia alternates between two dis t inct forms, the elementary replicating, Moulder, 1991). The infection begins with the EBs interacting with the host cell
14 membrane through electrostatic interactions. The EB then induces phagocytosis by the type three secretion system injecting the effector TARP (translocated actinrecruiting phosphoprotein) into the host cell, promoting actin remodeling and uptake (Clifton et al., 2004). Upon entry into the host cell, the EB quickly differentiates to the reticulate body (RB). While inside the host cell, Chlamydia resides within a vacuole termed an inclusion, which grows during the course of in fection. RBs replicate until an unknown signal causes the RBs to differentiate back to EBs asynchronously (Friis, 1972). Some RBs continue to replicate to produce RBs and EBs until lysis. The EBs are released and disseminate to initiate another round of infection (Moulder, 1991). The inclusion formed by Chlamydia allows it to reside within a privileged niche inside the host cell. Chlamydia alters the properties of the inclusion shortly after entry by insertion of proteins into the inclusion membrane. T hese membrane modifications ensure that the inclusion is not acidified by fusion with the endocytic or lysosomal pathway (Fields and Hackstadt, 2002). T o increase in size and acquire nutrients, the inclusion intercepts sphingomyelin and cholesterol from th e exocytic pathway (Hackstadt et al., 1995; Saka and Valdivia, 2010). Chlamydial proteins on the inclusion interact with microtubules in a dyneindependent and dynamitinindependent fashion (Grieshaber et al., 2003). The chlamydial inclusion migrates alo ng microtubules to the microtubuleorganizing center (MTOC). Migration to the MOTC is conserved among chlamydial species suggesting that MOTC localization is critical for the developmental cycle of Chlamydia The inclusion remains in close association wit h the MTOC throughout the host cell cycle and remains physically attached to centrosomes during mitosis (Grieshaber et al., 2006).
15 The obligate intracellular nature of the chlamydial infectious life cycle requires Chlamydia to reprogram many aspects of cellular function to render the cell hospitable to growth. Chlamydiae have a type III secretion system (T3SS) as wel l as an uncharacterized type II dependent secretion system. These secretion systems secrete protein effectors into the host cell, modulating c ellular behavior to create its unique intracellular niche (Zhong, 2011). One class of proteins that is translocated by the T3SS is the Inc proteins ( Inc lusion m embrane proteins), which comprise 7 10% of the proteome (Dehoux et al., 2011). The Inc proteins are inserted into the inclusion membrane and are characterized by a unique bilobal hydrophobic domain. Many of these Inc proteins also contain predicted coiled coil domains that suggest that Inc proteins interact with host proteins (Dehoux et al., 2011). Although Inc proteins are found in all Chlamydiae, specific Inc proteins are not well conserved (Read et al., 2000). Another way that Chlamydia manipulates its environment is through protease activity altering host protein levels. Chlamydia encodes approximately two dozen proteins with proteolytic activity (Stephens et al., 1998; Zhong, 2011). The most well studied chlamydial protease is the chlamydial proteaselike activity factor (CPAF), which is translocated to the host cytoplasm (Zhong et al., 2001; Shaw et al., 2002). CPAF is first translated as a proenzyme. Maturation occurs with dimerization and self proteolysis which regulates activity (Dong et al., 2004). Although true CPAF substrates are currently in question, CPAF is necessary for growth of C hlamydia and the inclusion (Jorgensen et al., 2011; Chen et al., 2012). Chlamydia infection induces a variety of cytopathic effects such as centrosome amplification, degradation of host proteins and multinucleation of the host cell (Greene
16 and Zhong, 2003; Grieshaber et al., 2006; Paschen et al., 2008; Johnson et al., 2009). Each of these defects can contribute to aneuploidy and chromosome instability in mammals (Chan, 2011; Weihua et al., 2011). Host Cell Chlamydial infection of dividing cells causes a nu mber of significant changes to the cellular pathways controlling cell division. Infection causes disorganized spindle assembly, early mitotic exit and amplification of centrosomes during G2 of the cell cycle (Johnson et al., 2009; Knowlton et al., 2011). Maintenance of cell cycle control is critical for normal cell division and is therefore a heavily regulated process Effects on the Cell Cycle To replicate the cell undergoes a series of events termed a cell cycle. The eukaryotic cell cycle is divided int o two periods: interphase, when the cell grows in size, and mitosis, the period when the cell divides the nuclear material. Interphase is further divided into three stages: Gap 1 (G1), Synthesis (S), and Gap 2 (G2) (Figure 1 1 ). G1 allows cells to determi ne if there are adequate nutrients available to begin the cell cycle (Menoyo et al., 2013). Furthermore, during G1 a cell determines if DNA damage has occurred during mitosis. If DNA damage has occurred, cells are delayed in G1 so that the damage can be r epaired before entry to S phase (Clmenson and Marsolier Kergoat, 2009). S phase is the portion of the cell cycle when chromosomes are replicated so the two forming daughter cells will receive identical complements of chromosomes. After S phase, the cell progresses into G2. During G2 the cell grows in size and again monitors for DNA damage (Clmenson and Marsolier Kergoat, 2009). If the cell is free of DNA damage, it will enter mitosis.
17 Chromosomes are physically divided during mitosis. This process is t ightly regulated to ensure that a symmetrical division occurs, allowing daughter cells to inherit the proper complement of chromosomes. Mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase (Figure 11 ). Prophase is characterized by chromosome condensation and nuclear envelope breakdown. Proph ase is followed by prometaphase during which the chromosomes are pushed and pulled by the microtubules of the mitotic spindle until aligned in the center of the cell (Wandke e t al., 2012). The cell has entered metaphase once the chromosomes align in the center of the cell termed the metaphase plate. Once chromosomes are aligned along the metaphase plate, the protein complex cohesin is cleaved allowing for sister chromosomes t o separate and enter anaphase (Uhlmann et al., 1999). Mitosis concludes with telophase in which the nuclear envelope reforms and chromosomes decondense. The cell cycle undergoes a unidirectional sequence due to a class of proteins termed cyclins whose lev els rise and fall causing progression through the phases. (Benanti, 2012). Cyclin expression is controlled through both transcrip tional regulation and ubiquitintargeted proteasome deg radation. Their function is to bind and activate cyclin dependent kinases (Cdks) whose activity is required for the cell to pass through the phases of the cell cycle (Pines, 2011). In order to enter S phase, Cyclin D1 must be expressed to activate Cdk4/6 (Jirawatnotai et al., 2012). Similarly, to enter mitosis Cyclin B1 bin ds and activates Cdk1, which facilitates nuclear envelope breakdown and chromosome condensation (Gavet and Pines, 2010).
18 Mitotic exit is controlled by the Spindle Assembly Checkpoint, which ensures that exit only occurs when a symmetrical mitotic spindle has formed. To satisfy the Spindle Assembly Checkpoint, all of the kinetochores must be attached to the mitotic spindle microtubules (Hartwell and Weinert, 1989). Once the checkpoint is satisfied the Anaphase Promoting Complex (APC) ubiquitinates securin and Cyclin B1, targeting them for degradation by the proteasome. Securin degradation releases its binding partner separase. Separase is a protease that degrades cohesin, the protein structure that forms a ring around sister chromosomes (Peters, 2006). T his cascade allows sister chromosomes to separate and the cell to proceed to anaphase. When Cyclin B1 is degraded, it inactivates its binding partner cyclin dependent kinase 1 (Cdk1). Inactivation of Cdk1 allows phosphatases to dephosphorylate targets of Cdk1 causing the cell to exit mitosis and reform the nuclear envelope (Baker et al., 2007). In order for the mitosis to result in two new daughter cells, the cell must undergo cytokinesis. Cytokinesis is the process of physically separating two cells. Cytokinesis begins during anaphase when the cell positions the actinmyosin contractile ring at the metaphase plate. The actinmyosin ring contracts to form a cleavage furrow followed by an intercellular bridge. smic connection between postmitotic sister cells (Steigemann and Gerlich, 2009b). The intercellular bridge contains tightly compacted microtubules and hundreds of proteins involved in the cytoskeleton, membrane traffic and signaling (Eggert et al., 2006). The inter cellular bridge is very proteindense and causes a barrier for diffusion between the two cells (Schmidt and Nichols, 2004). At the center of the intercellular bridge is a structure termed the midbody. The midbody is the area where antiparallel m icrotubules overlap
19 and are covered in a n electrondense matrix (Steigemann and Gerlich, 2009b). This structure provides an anchor for the ingressed cleavage furrow. The final step of cytokinesis is abscission, which is characterized by the resolution of the membrane between the postmitotic daughter cells (Glotzer, 2001; 2005). Both postmitotic daughter cells traffic exocytosed membrane to the midbody of the cell. Resolution of the intracellular bridge occurs when proteins have been cleared of the abscissi on point and new membrane has been delivered. Abscission cannot be reversed; therefore, a final checkpoint controls this crucial step. The NoCut pathway monitors for DNA in the midbody of the dividing cell, which occur due to DNA segregation errors. (Nord en et al., 2006) Upon activation, the NoCut pathway pauses the cell in cytokinesis to allow DNA an opportunity to be removed out of the midbody. DNA removal is important to prevent irreversible damage that would occur during abscission. If the DNA is not removed from the midbody, the cell will undergo cleavage furrow regression and abort cytokinesis resulting in a multinucleated cell ( Mendoza et al., 2009). The prevailing hypothesis for the fate of tetraploid cells is that they arrest in G1 due to p53 an d the arrest results in apoptosis or senescence of the cell (Andreassen et al., 2001). Recently, it has been found that some tetraploid cells with intact p53 continue to undergo the cell cycle suggesting that multinucleated cells are in fact viable (Uetake and Sluder, 2004). Tetraploid cells can lead to aneuploidy by two mechanisms: chromosome aberrations and extra centrosomes (Fujiwara et al., 2005; Fukasawa, 2007; Ganem and Pellman, 2007). It is unclear how tetraploid cells accumulate chromosomal breaks and
20 rearrangements, but one possibility is damage through a chaotic mitosis causing physical force on the chromosome. DNA damage is further promoted by extra centrosomes when they are not clustered into a pseudobipolar spindle (Ganem and Pellman, 2012). Finally, extra centrosomes promote missegregation of chromosomes by creating asymmetrical forces that act upon chromosomes. Effects on the Centrosome Cycle Centrosomes are nonmembranous organelles that form the microtubule organizing center (MTOC) which resides in a juxtanuclear position in eukaryotic cells (Gould and Borisy, 1977). A centrosome is composed of a mother and a daughter centriole that are crucial in anchoring microtubules and controlling the reproduction of centrosomes (Piel et al., 2000; Nigg, 2002). Each centriole is composed of nine sets of triplet microtubules and a centrosome is composed of two sister centrioles. Sister centrioles are arranged orthogonally and are surrounded by an electrondense cloud of proteins termed the pericentr iolar material (PCM) (Vogel et al., 1997). The PCM contains over 200 proteins, which creates a scaffolding and a nucleation site for microtubules (Andersen et al., 2003). In normal animal cells there are one to two centrosomes present depending on the phas e of the cell cycle (Figure 1 2). The cell undergoes a centrosome cycle to replicate the centrosome for mitosis. The centrosome cycle begins with untethering of the mother and daughter centrioles during anaphase (Kuriyama and Borisy, 1981). Separase, the same protease necessary for separation of sister chromatids, cleaves cohesin on sister centrioles and causes untethering (Schckel et al., 2011). Upon entry into S phase, the centrioles begin semi conservative replication with growth of new centrioles on the proximal part of the existing centriole (Kuriyama and Borisy, 1981). During G2 the centrosomes mature and
21 recruit additional PCM. Finally, the two centrosomes separate through removal of the tether at mitotic entry and the centrosome cycle begins again (Zyss and Gergely, 2009). The centrosome cycle and the cell cycle are tightly coupled to ensure there are only two centrosomes when the cell enters mitosis. Tight coupling is accomplished by using the same machinery to regulate both the cell cycle and the centrosome cycle. One way they are coupled is by the activation of cyclin dependent kinase Cdk2 that is necessary for both DNA replication and centrosome duplication, successfully preparing the cell for mitosis (Ferguson and Maller, 2010). Centroso me amplification occurs if the centrosome cycle is disturbed, the cell fails in cytokinesis, or fuses with another cell (Anderhub et al., 2012). In order to protect the cell from multipolar mitosis, eukaryotic cells have the innate ability to cluster extr a centrosomes during interphase and mitosis (Ring et al, 1982) If centrosome clustering is disrupted, it can promote cellular transformation. Cancer Biology Since the 1990s it has been recognized that infectious agents contribute to a substantial portion of cancers worldwide. Pisani et al. proposed 15.6% of cancers in 1990 were caused by viruses, bacteria or parasites (Pisani et al., 1997). More recently, de Martel et al. performed a study in which they took into account only infectious agents that the International Agency of Research (IAR) had classified as carcinogenic. This study found the burden to be 16.1% or around two million of the cancer cases in 2008 could be attributed to infectious agents (de Martel et al., 2012). Studies such as these do not take into account infectious agents that are considered cofactors, such as HIV, so the contribution of infection may be underestimated.
22 Studies have linked Chlamydia as a cofactor for increased cancer rates due to prior infection (Koskela et al., 2000; Anttila et al., 2001; Smith et al., 2002; Matsumoto et al., 2003; Smith et al., 2004; Madeleine et al., 2007; Roset Bahmanyar et al., 2012). Studies performed by Koskela et al. 2000 and Anttila et al. 2001 compared serum samples of 530,000 Nordic women who were either diagnosed with cervical cancer or the matched control. These studies demonstrated that patients who were seropositive for Chlamydia trachomatis had a twofold increased risk for squamous cell carcinoma (Koskela et al., 2000; Anttila et al., 2 001). A case study carried out by Smith et al. demonstrated that women seropositive for C. trachomatis and DNA positive for HPV had a twofold increase in squamous cervical cancer, confirming previous studies (Smith et al., 2002). Additional studies have used chlamydial DNA as a marker for past infections and have had similar results. Wallen et al. 2002 performed a populationbased study that examined samples from 130,000 women over a 26 year span. This study demonstrated a prior C. trachomatis infection was associated with an increased risk for the development of invasive cervical cancer (Wallin et al., 2002). Currently, the mechanism of how Chlamydia may contribute to cervical cancer is unknown. One hundred years ago, Theodor Boveri first proposed that malignancies might be a result of abnormal mitosis due to extra centrosomes (Boveri, 2008). His hypothesis was widely ignored after the discovery of genes and somatic mutations. Recently, due to the observation that most solid tumors have supernumerary ce ntrosomes, interest in the role of centrosome abnormalities in cellular transformation has increased (Brinkley and Goepfert, 1998; Pihan et al., 1998).
23 Although supernumerary centrosomes contribute to spindle multipolarity, extra centrosomes alone are insu fficient. Eukaryotic cells are able to suppress the effects of extra centrosomes in order to undergo bipolar mitoses (Ring et al., 1982; Brinkley, 2001; Quintyne et al., 2005). Many cells that undergo a multipolar mitosis instead of bipolar mitosis are i nviable due to substantial chromosome loss (Ganem et al., 2009) There are two ways that cells suppress the effects of amplified centrosomes to prevent multipolar spindles. The first is to functionally silence extra centrosomes preventing them from formi ng a spindle pole. Alternatively, cells have the ability to cluster extra centrosomes into two functional spindle poles (Nigg, 2002). Silencing and clustering extra centrosomes are not mutually exclusive and work together to transition from a multipolar s pindle in prometaphase to a pseudobipolar spindle in anaphase, allowing for viable daughter cells (Thompson et al, 2010) We have demonstrated that Chlamydia induces cytokinesis failure of the host cell. In the following chapters, we examine the effects chlamydial infection has on mitosis and centrosomes. We present evidence that the ability of Chlamydia trachomatis to override the Spindle Assembly Checkpoint, amplify centrosomes and decluster supernumerary centrosomes, causes DNA bridging and results i n cytokinesis failure. All three phenotypes are necessary for inducing cytokinesis failure; higher rates of declustered centrosomes in chlamydial species correlate to higher rates of multinucleation. We propose a model in which Chlamydia causes cellular t ransformation through induction of chromosomal instability.
24 Figure 11. The cell cycle. The eukaryotic cell cycle is divided into two periods: interphase, when the cell grows in size, and mitosis, the period when the cell divides the nuclear material. Interphase is further divided into three stages: Gap 1 (G1), Synthesis (S), and Gap 2 (G2). Mitosis is divided into five phases: prophase, prometaphase, metaphase, anaphase and telophase. The cyclins important for progressing through the cell cycle are noted on the inside of the cell cycle.
25 Figure 12. Centrosome Cycle. The centrosome cycle is a series of events that the cell undergoes in order to replicate centrosomes. Replication events coincide with replication events in the cell cycle. New centri oles form orthogonally to existing centrioles during S phase. The two centrosomes disengage during G2 in order to create two spindle poles during mitosis.
26 CHAPTER 2 MATERIALS AND METHODS Organisms and Cell Culture Chlamydia trachomatis serovar L2 (LGV 434) C. muriduram Nigg strain (referred to as MoPn), and C. caviae GPIC (gift from Ted Hackstadt) were grown in McCoy cells and EBs were purified by Renografin density gradient centrifugation as previously described (Caldwell et al., 1981). Coxiella burnetii Nine Mile phase II (NMII) clone 4 was a gift from Robert Heinzen, Rocky Mountain Labs, NIAID/NIH. All cell lines were obtained from the American Type Culture Collection. HeLa 229 cells and Neuroblastoma (N1E -115) cells were grown in RPMI 1640 (Cellgro) Foreskin Fibroblasts and McCoy cells were grown in DMEM (Cellgro) supplemented with Infections Cells were incubated with C. trachomatis C. muridarum or C. caviae EBs at an MOI of approximately 3 in Hanks balanced salt solution (HBSS) (Gibco) for 45 minutes at room temperature while rocking. The inoculum was removed and replaced with fresh RPMI 1640 For C. burnetii infections, the same procedure was used except the incubation was for 4 hours and no gentamicin was used. Immunohistochemistry Cells for fluorescent microscopy were grown on 12mm number 1.5 borosilicate glass coverslips coated with Poly L lysine (Sigma). For antibody staining, the coverslips were fixed in icecold methanol for 10 minutes and incubated with the primary antibody
27 described for each experiment. The antibodies used for these experiments were: mouse monoclonal anti tubulin (Sigma), mouse monoclonal anti tubulin (Sigma) and AlexaFluor 488 phalloidin (Invitrogen). C. trachomatis and C. burnetii were stained with platelet poor human serum (Sigma). To visualize the primary antibodies, AlexaFluor 488conjugated secondary antibodies against mouse IgG were used (Molecular Probes/Invitrogen). The far red fluorescent DNA dye DRAQ5 (Biostatus) or Hoechst was used to visualize nuclei. Images were acquired using a spinning disk confocal system connected to a Leica DMIRB microscope with a 63x oil immersion objective, equipped with a Photometrics cascadecooled EMCCD camera, under the control of the ( http://micro manager.org ). Images where processed with image analysis software ImageJ ( http://r sb.info.nih.gov/ij/ ). Images displayed for figures are maximal intensity projections of the 3D data sets, adjusted for brightness and contrast. Transfections and Plasmids HeLa cells were seeded on 25mm number 1.5 glass coverslips in 6well plates and gr own to 70% confluency. Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. A stable cell line of RFP H2B HeLa cells was and expression from the GFP vector was allowed to proceed for 48 hours to permit time for expression. Human H2B was cloned from pBOS H2BGFP (BD Biosciences) into a RFP destination vector using the Gateway Cloning System (Invitrogen) according to the manufacturers instruction. Similarly, Bicaudal D1 was cloned from pOTB7Bicaudal (ATCC) into a GFP destination vector using the Gateway Cloning System.
28 Live Cell Imaging Cells were transferred into high glucose DMEM media with 25 mM HEPES. The coverslip was overlayed with mineral oil and the chamber was supplemented with CO2. Images where acquired as previously described. Cell Fusion Assay HeLa cells were prestained with CytoTracker Red CMTPX or Green CMFDA (Molec ular Probes) using recommended protocol. Both populations of cells were infected with Chlamydia trachomatis at an MOI of 3. The two populations of cells were seeded together on 25 mm number 1.5 glass coverslips in 6well plates for 6 hpi Cells were fixed with cold methanol at 30 hpi and stained with Draq5. Alternatively, cells were pre stained and seeded without infection. The cells were then electroporated at 175V, and orange multinucleated cells were counted as positive for cell fusion. Inclusion Measurement HeLa cells were imaged using confocal microscopy. I mageJ was used to measure the area of the inclusion or CCV. We calculated and compared the 2 dimensional areas occupied by the CCV and inclusion to simplify quantification. In both the Coxiella CCV and chlamydial inclusion the Z component was comparable and did not significantly change over time. The flat cell shape highly constrained the shape of the vacuoles in the Z dimension, and significant growth only occurs in th e X and Y dimensions. Cell Free Degradation Assay Mitotic cell proteins were obtained through a mitotic shakeoff of HeLa cells in Phosphate Buffered Saline (PBS). Cells were pelleted and resuspended in 0.5 mL of
29 NP 40 Buffer (1% NP 40, 150 mM NaCl, 0.5% Triton X 100 in 50 mM Tris pH 8) Protease inhibitor cocktail (Sigma) was added. Extraction was carried out on ice for 20 minutes and then centrifuged to pellet the debris. Supernatant was collected and kept at 80 until use. Chlamydial proteins were collected through harvesting the cytosolic fraction of cells infected with C. trachomatis serovar L2. Cells were scraped and centrifuged to form a pellet. Cells were resuspended in a douncing buffer (10mM KCl, 1.5mM Mg Cl2, 1mM EDTA, 1mM DTT, 250mM sucrose in 20mM Hepes KOH pH 7.5). Cells were dounced, and the supernatants were harvested with a series of centrifugations with the final centrifugation at 100,000x g. CPAF peptide inhibitor (GenScript) was also added. For th e degradation assay, mitotic host proteins and chlamydial cytosolic proteins were mixed and incubated for 1 hour at 37 C Mitotic Index Assays The mitotic index was calculated by determining the ratio of mitotic cells to the total number of cells present. For the infected populations only infected cells were counted. A minimum of 1500 cells were counted over 2030 fields, and the procedure was repeated three times. The SAC was induced with the kinesin5 inhibitor monastrol (Sigma) and the mitotic index was again calculated. Cells were treated with monastrol cells were methanol fixed and stained with anti tubulin to visualize monopolar spindle formation, DRAQ5, and anti L2 antibodies where appropriate Western Blot Proteins from the cell free degradation assay were separated by SDS polyacrylamide gels and tr ansferred to nitrocellulose membranes. The primary antibodies were: mouse monoclonal anti cyclin B1, rabbit polyclonal anti cdc2, rabbit
30 polyclonal anti cdc2 (Thr161), (Cell Signaling Technolog y), rabbit polyclonal anti PTTG 1 (Invitrogen), mouse monoclonal anti GAPDH (EnCor). Proteins were visualized with either goat anti mouse perioxidase conjugated secondary antibodies (Thermo) or goat anti rabbit perioxidase conjugated secondary antibodies (Sigma) and SuperSignal West Dura Extended Duration Substrate (T hermo). Double Thymidine Block HeLa cells where grown in 2mM thymidine in RPMI for 18 hours. Cells were then switched to fresh RPMI for 8 hours followed by infection with Chlamydia as previously described. Immediately following the infection the cells wer e put back in 2 mM thymididne in RPMI. After 12 hours, the cells were released with fresh media and samples were collected at the times indicated. Fluorescence U biqui n it ation Cell Cy c le Indicator HeLa cells were transduced with PremoTM FUCCI Cell Cycle S ensor (Invitrogen) according to the manufacturers instructions. When necessary, the cells were infected with Chlamydia after the transduction. The cells were then imaged live for 18 hours, collecting frames every 10 minutes. For infected cells, this time period captures 1836 hours post infection. This experiment was repeated three times each for both uninfected and infected cell populations. Data sets were exported to an OMERO imaging data base ( http://www.openmicroscopy.org/site/products/omero). Omeros quantification tools were used for the live cell intensity analysis. Griseofulvin Treatment HeLa cells were infected as previously described for 16 hours. The media was then changed to incl ude 10 M griseofulvin (Sigma) for the subsequent 20 hours. Cells were fixed in ice cold methanol and stained.
31 Centrosome Calculations Centrosome to nuclei distance was calculated by using ImageJ to draw a line from the closest centrosome to the nucleus Centrosome spread was calculated by using 3D image stacks to produce a 2D binary image of the centrosomes. The 2D spread was calculated using the ImageJ plug in Hull and Circle. A region of interest was drawn around the centrosomes and the area of the bounding circle was calculated from the minimal fitted polygon. Statistical Analysis Numerical data are presented as the mean SEM, and were analyzed by either the unpaired t test or oneway ANOVA with a p<0.05 using GraphPad Prism4 software.
32 Table 21. Inhibitors used in experiments. Inhibitor Formula Action Cytochalasin D C 30 H 37 NO 6 Disrupts actin filaments, preventing actin polymerization Griseofulvin C 17 H1 7 CIO 6 At low concentrations declusters centrosomes Latrunculin A C 22 H 31 NO 5 S Binds monomeric actin, preventing actin polymerization Monastrol C 14 H 16 N 2 O 3 S Eg5 inhibitor that causes monopolar spindles during mitosis Table 22. Peptide Inhibitors Peptide Sequence MW Charge Purity CPAF inhibitor SEFYSPMVPHFWAELRNHYATSGLKS 3055.39 +2 >85% Scrambl ed inhibitor WNHSSTMGYFLLPAEVEKYSFSPHRA 3055.45 +2 >85%
33 CHAPTER 3 CHLAMYDIA INFECTION INDUCES HOST CYTOKINESIS FAILURE AT ABCISSION1 Introduction Chlamydia trachomatis is a major human pathogen with biovars that can cause both sexually transmitted disease (STD) and trachoma, the leading cause of infect ious blindness (Moulder, 1991; Hu et al., 2010) Infections caused by genital serovars of C. trachomatis can persist and lead to serious health sequelae such as pelvic inflammatory disease and tubal infertil ity in untreated women (Cates and Wasserheit, 1991). Chlamydia has also been clinically associated with cytologic cervical atypia (Kiviat et al., 1985) and epidemiologically linked to an increase in the risk for cervical cancer (Anttila et al., 2001; Smith et al., 2002; Madeleine et al., 2007). A direct molecular mechanism responsible for this link has yet to be described. The obligate intracellular nature of the chlamydial infectious life cycle requires Chlamydia to reprogram many aspects of cellular func tion to render the cell hospitable to growth. Chlamydiae have multiple systems to secrete protein effectors into the host, modulating cellular behavior to create its unique intracellular niche. Chlamydia expresses both a type III secretion system as well as an uncharacterized type II dependent secretion system to efficiently deliver toxins/effectors to the cel l to carry out these functions (Zhong, 2011; Dehoux:2011ep) These effectors induce a variety of cytopathic effects such as centrosome amplification, degradation of host proteins and multinucleation of the host cell (Greene and Zhong, 2003; Grieshaber et al., 2006; Paschen et al., 2008; Johnson et al., 2009). Multinucleated cells are common in all solid 1 Previously published as: Brown, H.M., Knowlton, A.E., and Grieshaber, S.S. (2012). Chlamydial Infection Induces Host Cytokinesis Failure at Abscission. Cell Microbiol.
34 tumors and contribute to aneuploidy and chromosome instability (Weihua et al., 2011eg) The induction of multinucleation of the host cell during chlamydial infection is a potential contributing factor in the observed increased risk of cancer in infected patients. Multinucleation can occur via cell fusion or cytokinesis failure, both of which have been shown to occur during viral infections. Disregulation of the cell cycle by viral infection leads to cytokinesis failure, and the membrane fusion events of budding viruses results in cell to cell fusion of neighboring cells (Duelli et al, 2007; Liu et al., 2005) Previous studies in Chlamydia suggest that either of these mechanisms could contribute to multinucleation. Egress of the chlamydial inclusion has been reported to be mediated, at least in part, by bud ding of the inclusion from the cell suggesting that Chlamydia may mediate membrane fusion events (KevinHybiske, 2007) Alternatively, we have recently determined that Chlamydia infection affects the cell cycle of the host cell by overriding the Spindle Ass embly Checkpoint (SAC) causing infected cells to prematurely exit mitosis (Knowlton et al., 2011). In this study we demonstrate that chlamydial induced multinucleation is entirely due to cytokinesis failure. Furthermore, we show that chlamydial induced mu ltinucleation occurs due to failure late in cytokinesis at the abscission step. Finally, we establish that Chlamydia proteaselike activity factor, CPAF, acts as an anaphase promoting complex by cleaving cyclin B1 and securin, allowing the chromosomes to p rematurely separate. This activity drives cells through metaphase before the chromosomes are properly aligned and attached, leading to lagging chromosomes. The lagging chromosomes persist in the midbody between the newly formed daughter cells and ultimatel y interfere with abscission.
35 Results Cytokinesis Fails Very Late in Infected Cells We previously reported that chlamydial infected cells proceed through mitosis faster than uninfected cells (Knowlton et al., 2011). To determine if this impacts cell divis ion, we directly examined infected dividing cells using live cell imaging. HeLa cells were transfected with GFP and infected with serovar L2. These cells were imaged every 10 minutes for 24 hours. Imaging was initiated at 20 hours post infection (hpi), as this is the start of the mature stage of the chlamydial infection. Images were collected for both infected and uninfected cells for a total of 41 infected cells and 39 uninfected cells that completed mitosis during imaging. The vast majority of the infecte d cells became multinucleated after mitosis (80.5% infected, Fig 31 C). This was a significant increase in multinucleation over the uninfected cell population (5.1% uninfected, Fig 3 1 C). In addition, cells were transfected with GFP Bicaudal D1 and imaged 24 hpi every 15 minutes for 18 hours. Bicaudal D1 was used to localize the chlamydial inclusion ( Matanis et al., 2002; Moorhead et al., 2007). We observed that multinucleation often occurred many hours after the completion of mitosis and in some cases oc curred after differential expression of the GFP Bicaudal D1 protein (Fig 3 1 A B). The observed differential GFP Bicaudal D1 expression between the apparent daughter cells that ultimately form a single multinucleated cell could be explained by two potential mechanisms; fusion of the closely associated cells after completion of cytokinesis or by failure late in the cytokinesis process after free diffusion is blocked by the formation of the midbody, a late cell division intermediate.
36 Multinucleation is Not Caused by Fusion The genital biovars of Chlamydia are reported to egress from the cell without lysis, suggesting chlamydial infection may induce cell membrane fusion events in a man ner similar to budding viruses (KevinHybiske, 2007; Todd and Caldwell, 1985) We employed a differential cell staining technique to determine if cell fusion occurs during infection. Two populations of cells were labeled with different fluorescent dyes and fusion events were scored by counting cells that fluoresced in both channel s. HeLa cells were intrinsically labeled with either CytoTracker green or CytoTracker red fluorescent dyes. These two cell populations were then independently infected with Chlamydia L2. The two cell populations were trypsinized and coplated onto coversli ps 6 hpi and allowed to incubate for a total of 36 hours. These experiments were repeated with the genital serovar G but incubated for 48 hours, due to the slower growth rate. As a control to verify our ability to detect multinucleation, two uninfected cel l populations were labeled with CytoTracker red or CytoTracker green, coplated and subjected to an electrical current to induce fusion. Yellow stained cells indicating fusion between red and green dyed cells were enumerated at the indicated time points. N o fusion could be detected in either serovars L2 or G infected samples with greater than 1000 multinucleated cells counted (Fig 3 1 D). Conversely, yellow stained cells indicating cell fusion were readily evident in the electrofused samples. These data rule out fusion as a contributing mechanism for multinucleation.
37 Cytokinesis Fails After Ingression Upon ruling out fusion as a mechanism for multinucleation, we sought to determine at which step cytokinesis fails in the chlamydial infected cells. Cytokinesis can be divided into multiple stages; specification of the cleavage plane and ingression of the cleavage furrow, followed by formation of the midbody and abscission (Normand and King,2010) Ingression of the cleavage furrow initiates with the formation of an actin myosin ring during late anaphase in order to mechanically separate the daughter cells. To morphologically assess the structural organization of the contractile ring, infected cells were stained with fluorescently labeled phalloidin. The actin organization at the cleavage furrow appeared to be unperturbed by the chlamydial inclusion as there was no significant observable differences between infected and uninfected dividing cells (Fig 3 2A). As an additional line of evidence that the actin cleavage furrow was not disrupted during infection, we compared infected cells to those treated with the actin depolymerizing compounds cyctochalasin D or latrunculin A. Disruption of the actin contractile ring during cytokinesis results in multinucleation and i mmediate arrest in G1 of the cell cycle (Lohez et al., 2003) Centrosome amplification induced by chlamydial infection is dependent on progression of the cell cycle into G2 (Johnson et al., 2009). We used this observation to assess the effects of cytokines is failure induced by Chlamydia on cell cycle progression. As a measure of cell cycle progression we calculated the number of centrosomes per cell. The infected cells not treated with the actin depolymerizing drugs failed cytokinesis and had a clear increa se in centrosome numbers (average centrosomes per cell 2.06, Fig 3 2B). However, infected cells treated with either cytochalasin D or latrunculin A became multinucleated but had no increase in
38 centrosome numbers over treated uninfected cells (average centr osome per cell 1.71, Fig. 3 2 B). The observation that the centrosome phenotype (no amplification) induced by disruption of the actin contractile ring was dominant over the chlamydial induced phenotype (amplification) demonstrates that chlamydial induced c ytokinesis failure occurs at a step after formation and function of the contractile ring. In addition to the lack of disruption of the actin cleavage furrow, the invaginated region of the cell did not appear to be sterically hindered by the chlamydial inc lusion. To specifically address the effects of steric interference on cell division, we compared the multinucleation rate in cells infected with Coxiella burnetii with cells infected with C. trachomatis L2. C. burnetii is a bacterial pathogen with a eukary otic intracellular life cycle that takes place in a vacuole termed the Coxiella containing vacuole (CCV) (Heinzen et al.,1999) Although the growth rate and characteristics of the intracellular niche differ between C. trachomatis and Coxiella, both organis ms construct and maintain a large intracellular parasitophorous vacuole in which replication occurs (Heinzen et al.,1996) We measured the multinucleation rate of cells infected with C trachomatis L2 and in agreement with previous reports, multinucleation is significant by 18 hpi (Fig 2D). The size of the inclusion at these early time points is similar to the size of the CCV at 96 hours due to Coxiellas slower growth rate. Cells infected with Coxiella for 96 hours displayed no increase in multinucleation compared to uninfected cells (Fig 3 2C and D). To specifically compare the steric effects of CCVs that were of similar size to the chlamydial inclusions, we measured the size of inclusions in chlamydial infected multinucleated cells at 18 hpi (average size of 5,588 m2450). We then calculated the multinucleation rate in Coxiella infected cells containing CCVs of comparable size
39 (average size of 6,429 m2350, Fig 3 2C). There was no increase in multinucleation in this population of cells over uninfected cells. This demonstrates that simple steric effects exerted by the presence of the chlamydial inclusion does not account for the rate of cytokinesis failure observed. The lack of cell cycle arrest, correct formation of the contractile ring, lack of steric i nterference from the chlamydial inclusion and the inhibition of diffusion prior to multinucleation all suggest that cytokinesis failure is occurring very late, likely at abscission. Infected Dividing Cells Make Midbodies Following formation of the actinmy osin contractile ring, cleavage furrow formation and ingression, the spindle microtubules are compacted to form an intracellular bridge containing densely packed microtubules termed the midbody (Caballe and MartinSerrano, 2011). Abscission occurs after resolution of the membrane within the midbody to form the final independent daughter cells (Caballe and Martin Serrano, 2011). To visualize the progression of cytokinesis, chlamydial infected cells were stained with anti tubulin allowing us to quantify the effect of infection on the formation of the midbody, the last step before completion of cytokinesis. In uninfected HeLa cells, 74.2 4.6% of post metaphase mitotic cells possessed fully formed midbodies. This number increased to 92.2 0.6% in the 36 hour chlamydial infected cell population (Fig 3 3A and B). UV treatment of uninfected cells was used as a control as UV treatment causes failure in cytokinesis by inducing DNA cross bridging and forcing cells to abort cytokinesis at the abscission stage (Norden et al., 2006). We found that midbodies were present in 83.0 1.2% of post metaphase cells post UV exposure (Fig 3 3 B) indicating that cells that fail cytokinesis due to DNA bridges successfully form
40 midbodies. We also assessed midbody formation in cel ls that failed cytokinesis due to failure in the function of the actin contractile ring using cytochalasin D and latrunculin. These cells never progressed to midbody formation (Data not shown). The proper formation of midbodies during infection further indicates that chlamydial infected cells successfully proceeded through the actinmyosin contractile ring stage into this late stage of cytokinesis. The observation that chlamydial infection increased the number of cells with intact midbodies also suggested t hat there is a delay in the resolution of the midbody and that infected cells fail in cytokinesis at this stage. This is further supported by the observed differential expression of GFP observed in infected dividing cells because diffusion is limited by the c ompact midbody structure (Fig 3 1 ). Lagging Chromosomes are Increased in Frequency in the Midbody of Infected Cells and Correlate with Multinucleation Previous studies from our lab demonstrated that infected cells prematurely exit mitosis (Knowlton et al., 2011). A cell that exits mitosis before metaphase is complete can acquire chromosome segregation defects. These defects are commonly associated with cytokinesis failure as chromosome segregation errors can lead to chromosome nondisjunction and laggin g chromosomes that bridge the midbody (King, 2008) We therefore examined dividing cells for the presence of DNA between the forming daughter cells. Cells infected with serovar L2 for 32 hours and uninfected cells were stained for DNA, microtubules and Chlamydia (Fig 3 4 A). Mitotic cells in these populations were quantified for the presence of DNA in the midbody between the two daughter cells. The infected cell population had a significant increase in the number of cells containing DNA in the midbody compared to uninfected (26.3% and 6.7% respectively, Fig 3 4B). Due to chromosome decondensation during mitotic exit limiting
41 detection, the number of cells with DNA in the midbody may be higher (Steigemann et al., 2009). In order to confirm that DNA in the midbody resulted in cytokinesis failure, we performed live cell imaging (Fig 3 4C). Uninfected cells succeed in mitosis 94.8% of the time, correlating with the low level of multinucleation observed in HeLa cells (Fig 3 4D). Surprisingly, we observed that unin fected cells displayed some level of DNA disorganization after metaphase. In this population 41.0% of successful cytokinesis events had evidence of DNA disorganization. The number of uninfected cells that failed cytokinesis was small (5.1%), however only half of these cells that failed cytokinesis had evidence of DNA disorganization suggesting that cytokinesis failure in uninfected cells was rarely caused by the presence of DNA in the midbody. In contrast, the rate of cytokinesis failure in the infected cel l population rose to 80.5% with 93.9% of these demonstrating lagging DNA between daughter cells (Fig 3 4D). These data further suggest that the increase in lagging chromosomes and chromosome nondisjunction directly contributes to chlamydial induced cy toki nesis failure. Chlamydial Infected Cells do not Arrest in Metaphase The spindle assembly checkpoint (SAC) regulates the timing of mitotic exit. This checkpoint blocks the progression at the metaphase to anaphase transition until chromosomes are properly al igned and have obtained bipolar attachment (McLean et al., 2011) This helps guarantee that the chromosomes are properly segregated to the poles and allows the chromosomes to be cleared from the cleavage furrow. As chlamydial infection increased the incidence of chromosomes found in the midbody between mitotic cells, we asked whether infection affects the SAC. To determine if Chlamydia infection is affecting the SAC, we triggered mitotic arrest with the drug monastrol. Monastrol specifically inhibits kinesin Eg5 resulting in the f ormation of
42 monopolar spindles (Kapoor and Mitchison, 2001) The disruption of bipolar spindle triggers the SAC, resulting in mitotic arrest in metaphase (Kapoor and Mitchison, 2001). Hela cells were infected with C. trachomatis sero var L2 and treated with monastrol for 24 hours. To assay for induction of the SAC, the mitotic index was measured by counting the percentage of cells in mitosis. Mitotic cells were stained with Hoechst to visualize DNA. The uninfected population had a mitotic index of 6.6 0.3% which decreased to 4.4 0.1% after infection (Fig 3 5A). Monastrol treatment led to an increase to 27.0 1.1% and again when infected with Chlamydia this decreased to 21.9 0.6%. These decreases showed statistical significance (p<0.003 and p<0.01, respectively). HeLa cells are a transformed cancer cell line and likely have changes in cell cycle check point control. Thus, we also looked at the effects of chlamydial infection on the SAC in a primary cell line. The mitotic index of primary human foreskin fibroblasts was 2.9 0.4% which dropped to 0.3 0.1% when infected with C. trachomatis L2 (Fig 3 5B). Treatment of the fibroblasts with monastrol resulted in an increase of the mitotic index to 21.4 0.8% indicating mitotic arrest. Monastrol treated infected primary fibroblasts showed a decrease in the mitotic index. The index dropped to 1.9 0.1% suggesting that these cells no longer arrested in mitosis. Although fibroblasts are not a typical target cell of a natural chlamydial inf ectio n cell cycle control in all non transformed cells is regulated by the same highly conserved checkpoint proteins. These data together demonstrate that infected cells do not arrest as efficiently during mitosis, even when the SAC is triggered. We previ ously demonstrated that cyclin B1 and securin were degraded during chlamydial infection (Knowlton et al., 2011). Both of these proteins are important
43 regulatory components of the SAC, which controls mitotic exit. (Kim and Yu, 2011; Pines, 2011; van Leuken et al., 2008) The secreted chlamydial protease CPAF has been reported to degrade cyclin B1 (Balsara et al., 2006; Paschen et al., 2008). We reasoned that CPAF may be acting as an anaphase promoting complex mimic, causing premature exit from mitosis. The an aphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase and regulates the SAC by targeting key molecules for degradation by the proteosome. (Baker et al., 2007; van Leuken et al., 2008) To test the hypothesis that CPAF is the chlamydial factor act ing as an APC/C we purified a crude cytosolic fraction from chlamydial infected cells and tested the ability of this fraction to degrade cyclin B1 and securin. This fraction contains the active CPAF molecule that is secreted from Chlamydia (Huang et al., 2008). We analyzed the degradation of both cyclin B1 and securin by western blot analysis. Lysates from chlamydial infected cells but not uninfected cells resulted in degradation of both cyclin B1 and securin purified from mitotic uninfected cells (Fig 3 6A ). We tested whether this degradation was due to CPAF proteolytic activity by using a specific CPAF inhibitory peptide. This peptide is derived from a self inhibitory fragment of the CPAF zymogen that blocks access to the substrate binding pocket as descri bed by Huang et al (Huang et al., 2008). This inhibitory peptide was demonstrated to have high potency against CPAF and is very specific as it does not affect the proteosome or CPAF from the closely related bacteria C. caviae (Jorgensen et al, 2011). Weste rn blotting revealed that only infected lysates could actively degrade cyclin B1 and securin. This degradation could be inhibited by the CPAF inhibitory peptide, but a scrambled peptide did not block degradation. Furthermore, treatment of infected lysates with a protease inhibitor cocktail did not
44 inhibit degradation (Fig 3 6B), confirming that host proteases were not responsible for the degradation of cyclin B1 and securin. To test whether CPAF mediated degradation of cyclin B1 resulted in its loss of func tion we assayed for the activity of cyclin dependent kinase 1 (Cdk1). Cdk1 is active when bound to cyclin B1 and becomes phosphorylated on T161. We assessed the activity state of Cdk1 in infected mitotic cells using an antibody specific for this species. W estern blotting revealed a drastic reduction in levels of phosphorylated active Cdk1 in chlamydial infected cells, supporting the hypothesis that degradation of cyclin B1 results in inactivation of the SAC (Fig 3 6C). Cyclin B1 Cdk1 must accumulate and become active in order for the cell to enter m itosis (Gavet and Pines, 2010 ; Marangos and Carroll, 2008). To determine if degradation of cyclin B1 by CPAF alters the host cells ability to enter mitosis we performed a mitotic index at various points after a thymidine release. Both uninfected and infected cells began to enter mitosis at 10 hour post release, but infected cells do not undergo a robust entry into mitosis (Fig 3 7A). Instead, infected cells show larger variability in the time it takes them to ent er mitosis as is shown by the shallower slope on the graph (Fig 3 7A). Uninfected cells had a peek of mitotic entry at about 12 hours after release from the block. In comparison, the chlamydial infected cells still had not reached a peak of mitotic entry by 13 hours after block release. This delay tracks with cyclin B1 levels in these two populations. The uninfected cell population showed a larger and faster increase in cyclin B1 levels after the block then did the infected cells (Fig 3 7B). Additionally, w e examined the length of S/G2 in uninfected and infected cells usi ng the FUCCI cell cycle sensor ( SakaueSawano et al ., 200 8 ) HeLa cells were transduced with Premo FUCCI Cell Cycle Sensor and then infected with Chlamydia
45 The length of S/G2 was calculat ed by measuring the time from the degradation of Cdt1RFP (degraded at the beginning of S phase) to nuclear membrane breakdown. We determined that in uninfected cells S/G2 was 507.3 78.4 minutes, while infected cells had a longer S/G2 that lasted 774.6 6 8.7 minutes (Fig 3 7C). Together these data demonstrate that the length of S/G2 is altered in infected cells and that this delay in entering mitosis correlates with a sl ower accumulation of cyclin B1. Discussion Multinucleation has been reported during chl amydial infections of cultured cells previously by Greene et al. and Sun et al. (Greene and Zhong, 2003; Sun et al., 2011). In this study we demonstrated that multinucleation during chlamydial infection is entirely caused by failure in cytokinesis and, in many cases, this failure occurs hours after apparent separation of the two daughter cells. By careful examination of the stages of cytokinesis in infected cells, we were able to determine that infected cells progressed through actinmyosin contractile ring formation and formed the typical condensed microtubule midbody structure that precedes the final stages of cell division, abscission. It is during this final stage that cytokinesis fails and multinucleation occurs. Abscission is irreversible for cytokines is and is therefore regulated by a variety of pathways. By observing live infected cells by fluorescent microscopy we demonstrated that the population of cells failing cytokinesis were those that had DNA segregation errors caused by chlamydial infection. T he presence of DNA in the midbody of dividing cells is a trigger to the cell to abort cytokinesis (Norden et al., 2006; Steigemann and Gerlich, 2009b). Our evidence supports the hypothesis that induction of DNA segregation errors during cytokinesis is a si gnificant factor leading to cytokinesis failure in chlamydial infected cells. We demonstrated in previous studies that chlamydial infection shortens
46 mitosis of the host cell, causing a disorganized metaphase to anaphase transition (Knowlton et al., 2011). In this study we were able to connect the observed high rate of multinucleation to the induction of disorganized mitosis. During chlamydial infection both cyclin B1 and securin are degraded reducing the time dividing cells spend in metaphase (Knowlton et al., 2011). Here, we extended this observation demonstrating that the chlamydial toxin CPAF acts as an anaphase promoting factor mimic, directly degrading cyclin B1 and securin resulting in the inactivation of Cdk1. This activity is likely responsible for overriding the SAC allowing cells to proceed to anaphase with disorganized chromosomes, resulting in DNA segregation errors. A recent study from the Harrison lab suggested a role for steric effects of the chlamydial inclusion leading to asymmetric cleavage furrow formation (Sun et al., 2011). In our studies we focused on events midway through the infectious cycle, before the chlamydial inclusion became the majority of the cell volume. We directly compared the effects of Coxiella vacuoles of equal or gr e ater size than those formed in chlamydial infected cells. In cells with large Coxiella vacuoles there was no increase in multinucleation, demonstrating that the presence of a large inclusion body is insufficient to cause multinucleation. It is possible that when the inclusion increases in size that steric effects also contribute to cytokinesis failure. Our results indicate that early exit from metaphase, caused by CPAF activity, is capable of causing lagging chromosomes that inhibit cytokinesis. However it is possible other chlamydial factors also contribute to cytokinesis failure. The I nc proteins CT223 and to a lesser extent CT224 and CT225 cause cytokinesis failure when ectopically expressed in mammalian cells (Alzhanov et al., 2009). The mechanism by which they
47 contribute to this failure is not known and ectopic expression is not always a reliable assay to assess the function of bacterial proteins. Cyclin B1 and activated Cdk1 are required for cells to enter mitosis leading to the question of how degradation of cyclin B1 during the chlamydial infection affects entry into mitosis. In this study we show that infected cells have a delay in mitotic entry after S phase arrest and spend more time in S/G2 then uninfected cells. Cyclin B1 is synthesised in the cytosol but is shuttled into the nucleus. In the nucleus cyclin B1 and its partner active Cdk1 reach a threshold level triggering mitosis. We speculate that during chlamydial infection cyclin B1 levels are reduced as CPAF targets it for destruction in the cytos ol, however, after cyclin B1 enters the nucleus it is protected from the activity of CPAF. This would explain the delay in mitotic entry. After NMBD cyclin B1 again becomes exposed to CPAF and is degraded pushing cells out of mitosis prematurely. Ov er all, our data support a model where the combined cytopathic effects of chlamydial infection subsequently lead to multinucleation. In this model chlamydial infection leads to centrosome amplification and declustering of centrosomes (Grieshaber et al., 2006; J ohnson et al., 2009; Knowlton et al., 2011). Additionally, CPAF degrades cyclin B1 and securin during mitosis leading to deactivation of Cdk1 and early exit from metaphase. Because of centrosome and spindle defects, the DNA is never properly aligned during metaphase leading to segregation errors during cytokinesis. These events ultimately lead to DNA bridging between daughter cells causing abscission failure (Fig 38). By inducing aneuploidy through lagging
48 chromosomes and cytokinesis failure, infection wit h Chlamydia may contribute to cellular transformation and cervical cancer. The target of a naturally occurring chlamydial infection is the epithelial cell layer. These cells are highly differentiated and are reported to have a low populat ion of replicatin g cells (Ramos et al., 2002) We speculate that the infection of replicating cells during a natural infection is a rare occurrence and therefore the induction of multinucleation is uncommon. However, HPV infection can drive cells out of quiescence and caus e cells to reenter the cell cycle (Korzeniewski et al.,2011; McLaughlinDrubin and Munger, 2009) We theorize that this population of cells would be more sensitive to cell cycle effects caused by chlamydial infection during a coinfection occurrence.
49 Figure 31. Multinucleation of host c ells is due to cytokinesis f ailure and not cell fusion. A) HeLa cells were transfected with GFP bicaudal D1 and then infected with C. trachomatis L2 for 24h. The cells were then i maged every 15 min for 18h. Images are time points from live cell imaging. Time 0 is started at telophase of the cell. Stars mark nuclei while the arrow points to the i nclusion. GFP signal was graphed with an intensity plot to allow visual comparison of differing intensities between forming daughter cells. B) Live cell imaging was performed on Chlamydia infected HeLa cells. Beginning at 20 hpi cells were imaged every 10 minutes for 24 hours. Uninfected mitotic cells resulted in multinucleation in 5.1% of the cells, while infected mitotic cells became multinucleated 80.5%. n=39 and n=41, respectively. C) Two different populations of HeLa cells were stained with Cytotracker Red or Green. Cells were coplated and either electroporated or infected with C. trachomatis L2 or G. Electroporation caused fusion as shown by the yellow multinucleated cell. Neither serovars of C. trachomatis induced fusion. 1000 multinucleated cells were counted and all multinucleated cells were due to cytokinesis failure.
50 Figure 32. Chlamydia does not sterically hinder cytokinesis. A) HeLa cells were infected for 30 hours then fixed and imaged using immunofluorescence. Cells were stained with either phalloidin (green), Draq 5 for DNA (blue) and human serum for Chlamydia (red). The actin ring was able to properly form i n Chlamydia infected cells. A rrows point to the actin ring. B) HeLa cells were treated with cytochalasin D or latrunculin A and the centrosome to nuclei ratio was evaluated using anti tubulin and Draq5. Uninfected cells and infected cells treated with cy tochalasin D did not show a significant difference in centrosome to nuclei ratio, having a ratio of 1.7 0.1 and 1.7 0.1 respectively. Similarly, when cells were treated with latrunculin A, uninfected cells had a centrosome to nuclei ratio of 1.6 0.1, while infected cells had a ratio of 1.7 0.1 Untreated infected cells showed a significant increase with an average of 2.1 0.1 centrosomes per cell p = 0.001. N= 3 experiments with at le ast 150 nuclei per experiment. C) Cells were infected with Chlamyd ia for 18 hours or Coxiella for 96 hours, then fixed. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). The arrow indi cates the location of the CCV. D) Cells were infected with Chlamydia or Coxiella for the indicated amounts of time and percent multinucleation of the population of infected cells were counted. Uninfected HeLa had a multinucleation rate of 2.9 0.3% while infection increased multinucleation with time. The infected rates were 8.02 0.81%, 8.26 0.50%, 13.2 1.3%, 17.2 0.5% for 18, 20, 22, 24 hpi respectively. All chlamydial time points investigated demonstrated a significance of p < 0.005. Coxiella had a multinucleation rate of 3.4 0.2%. N=3 experiments with at least 600 cells per experiment.
51 Figur e 33. Chlamydia infected cells form midbodies. A B ) HeLa cells were infected for 30 hours then fixed and imaged using immunofluorescence. Cells were stained with anti tubulin (green), human serum (red), and Draq 5 (blue). Arrows indicate location of the midbody. B ) All cells that were post metaphase in mitosis were evaluated for midbody formation. Midbodies were present in 74.2 4.6 % of uninfected cells exiting mitosis, while this percentage rose to 92.2 0.6% in cells infected with Chlamydia. A midbo dy was formed in 83.0 1.2% of cells exiting mitosis that were exposed to UV light. Infected cells demonstrated a significant increase in midbody formation with a p=0.017. N= 3 experiments with at least 150 cells per experiment.
52 Figure 34. Chlamydi a induces lagging chromosomes in infected cells. A) HeLa cells were infected for 30 hours then fixed and imaged. Conversely, uninfected cells were exposed to UV light for 3 minutes, allowed to grow for 24 hours and then fixed. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue) then examined in late telophase. Arrows indicate DNA bridging. B) Uninfected cells had DNA in the midbody 6.7 1.5% of the time, while infected cells had DNA in the midbody 26.3 1.2% of the time. Wh en cells were exposed to UV light there was DNA present in the midbody 19.00
53 1.5% of the time. N=3 experiments with at least 200 cells per experiment. A student t test was performed with p<0.02. C) Stable RFP H2B HeLa cells were transfected with GFP and i nfected with L2 for 20 hours. Cells were then imaged every 10 minutes for 24 hours. Panels show metaphase at time 0, followed by anaphase second and finally telophase of uninfected and infected cells. D) Cells were followed through mitosis with live cell i maging as shown in C. Cells were scored based on if they failed or succeeded in mitosis along with if DNA missegregation events were present. In uninfected cells, 94.9% of cells had successful mitosis, while only 19.5% of infected cells were successful. N= 39 and N=41 cells, respectively.
54 Figure 35. Chlamydia infection causes the premature exit of mitosis. HeLa cells were fixed 30 hpi and stained with anti tubulin (green), human serum (red), and Draq5 (blue). Mitotic cells were compared to the total number of cells after infection and/or monastrol treatment. N= 3 experiments with at le ast 1500 cells per ex periment. A) Uninfected HeLa cells had a mitotic index of 6.6 0.3% which decreased to 4.4 0.1% when infected, with a significance of p=0.003. When treated with monastrol 27.0 1.1% of uninfected HeLa cells arrest in mitosis, but only 21.9 0.6% of in fected cells, p<0.01. B) Fibroblasts had a mitotic index of 2.9 0.4% and 0.3 0.1% f or uninfected and infected cells, respectively, with a significance of p<0.002. Monastrol treated uninfected fibroblasts had a mitotic index of 21.4 0.8%, while infect ed fibroblasts had a mitotic index of 1.9 0.03%, with p<0.0001.
55 Figure 36. CPAF degrades cyclin B1 and securin. A) A cell free degradation was performed and analyzed by western blot. Cyclin B1 and securin were degraded by infected cell lysate. Degr adation could be blocked with a sm all peptide inhibitor of CPAF. B) Same experiment as A, but infected cell lysate was treated with a protease inhibitor cocktail. C) Mitotic shake offs were performed to harvest proteins from uninfected and infected cells. Western blots show that the level of Cdk1 is unchanged due to infection, but the active T161 form of Cdk1 is not present in infected cells.
56 Figure 37. Host cell entry into mitosis A B) A double thymidine block and infection was performed on HeLa cel ls. Cells were released and samples were collected at time points indicated. A) Cells were fixed and stained for anti tubulin (green), human serum (red), and Draq5 (blue) and a mitotic index was performed. In uninfected cells, a significant increase in mitotic cel ls begins at 10 hours post release, with the peak of mitosis occuring 12 hours after release from thymidine. Infected cells begin entering mitosis at 10 hours as well, but are not tightly synchronized. B C ) Western blot analysis was performed on uninfected and infected cell lysates. Cyclin B1 levels were evaluat ed for cells entering mitosis. D ) HeLa cells were transduced using the FUCCI system and infected with Chlamydia Starting 18 hpi, the cells were imaged every 10 minutes for 18 hours. The duration of S/G2 was calculated from the time of Cdt1 RFP degredation to mitotic entry (nuclear membrane breakdown). Uninfected cells were in S/G2 for 507 78.4 minutes while infected required 774.6 68.7 minutes with N=11, 13 respectively. The duration was signific antly different with p<0.02.
57 Figure 38. Model of chlamydial induced multinucleation. Chlamydial inclusions (red) secrete CPAF into the cytoplasm of the host cell (yellow). CPAF mimics the APC causing the cell to exit metaphase before microtubules (black) attach to the kinetochores of the chromosomes (blue). Furthermore, premature exit from metaphase does not allow supernumerary centrosomes (green) to cluster. This results in lagging chromosomes that become trapped in the midbody of the host cell. The host cell aborts abscission via the No Cut Pathway in order to protect the DNA. Lagging chromosomes can result in micronuclei.
58 CHAPTER 4 DECLUSTERING OF HOST CELL CENTROSOMES IS NOT CONSERVED AMONG SPECIES OF CHLAMYDIA Introduction Chlamydia trachomatis is an obligate intracellular pathogen that is the causative agent of the sexually transmitted infection (STI) Chlamydia (Moulder, 1991). Chlamydia is the most common bacterial STI with an estimated 4 million cases annually in the United States (Belland et al., 2004). Furthermore, C. trachomatis is of significant clinical importance because infection can result in pelvic inflammatory disease, ectopic pregnancy, and infertility (Cates and Wasserheit, 1991). More recently, C. trachomatis has been epidemi ologically linked to cervical and ovarian cancer (Koskela et al., 2000; Anttila et al., 2001; Alibek et al., 2012). Chlamydial infection overrides the Spindle Assembly Checkpoint (SAC), amplifies centrosomes and prevent s clustering of the centrosomes (Grieshaber et al., 2006; Johnson et al., 2009; Knowlton et al., 2011; Brown et al., 2012). Additionally, we demonstrated that these phenotypes may act together to cause multinucleation of infected cells (Brown et al., 2012). Multinucleated cells are common in all solid tumors and contribute to aneuploidy and chromosome instability (Weihua et al., 2011). The chlamydial factors involved in the induction of these phenotypes leading to multinucleation and aneuploidy are currently unknown. Therefore, we investi gated the ubiquity of the induction of these phenotypes across divergent chlamydial species. We compared the ability of C. trachomatis L2, C. muridarum (MoPn) and C. caviae (GPIC) in their ability to induce centrosome amplification. C. muridarum is a mouse specific pathogen that is evolutionarily closely related to C. trachomatis. Its genome has been sequenced, and 98.7% of the genes are conserved between C. trachomatis and C.
59 muridarum (Read et al., 2003). C. muridarum is well studied and used as the mouse model for human genital tract infection. GPIC was chosen because it is more distantly related to C. trachomatis, with 91.1% of genes conserved between the species (Read et al., 2003). GPIC is closely related to C. pneumoniae, a human pathogen of th e respiratory tract. Guinea pig infection with GPIC is the best animal model for human C. pneumoniae infection. All three species are well studied and grow at a similar rate, requiring approximately 48 hours to complete infection. T herefore these species are easy to compare at the same developmental stage. Although the genus Chlamydia has a large host diversity, wide tissue tropism and varied disease pathology, there is a high conservation of genes among the different species. GPIC, MoPn and C. trachomat is have 810 genes conserved between all three (Read et al., 2003). Despite having highly conserved genes, there is variation in expression of genes between species. For example, despite the fact that GPIC contains an identical set of glycogenmetabolizing genes, GPIC does not accumulate glycogen like C. trachomatis and C. muridarum (Moulder, 1991; Iliffe Lee and McClarty, 2000) Another variation among Chlamydia sp p. is in their Inc proteins ( Inc lusion membrane proteins), which are a vital part of chlamydi al host interactions due to their localization to the inclusion. C. trachomatis has 20 confirmed Inc proteins, but only a small subset is conserved among other species (Dehoux et al., 2011). Finally, the appearance of the inclusion varies between species. Phenotypically, the L2 and MoPn inclusion s form a smooth sphere, while GPIC forms a multi lobed, raspberry like structure.
60 In this study we demonstrate that changes in the host cell due to Chlamydia infection is not conserved among species. We found that all three species caused the same rate of centrosome amplification and prematurely exit mitosis, but C. trachomatis had the highest rate of centrosome declustering that correlated with rates of multinucleation. Results Chlamydia Species Cause Different R ates of Multinucleation C. trachomatis causes multinucleation through cytokinesis failure in a majority of the cells that undergo mitosis (Greene and Zhong, 2003; Brown et al., 2012). In order to determine if this phenotype was conserved throughout the Ch lamydia genus, we determined the multinucleation rate of the closely related species C. muridarum (MoPn) and the more divergent C. caviae (GPIC). Uninfected cells have a multinucleation rate of 2.9 1.0%. We determined that at 40 hours post infection (hpi) GPIC and MoPn cause multinucleation rates of 10.9 0.9 % and 28.1 0.6%, respectively. These multinucleation rates are significantly lower than the 64.7 2.5% that is observed in L2infected cells ( Figure 4 1B) All three species have similar growt h rates, but to ensure our findings were not due to differences in inclusion size we measured the area of the inclusions. The inclusion sizes for GPIC, MoPn and L2 were 328 93 2, 360 122 2, and 351 139 2 respectively. The inclusion sizes were not significantly different with a pvalue of >0.3. We aimed to determine the differences in host pathogen interactions between species from these findings. Our previous studies suggested that multinucleation results from a combination of changes to the cells that cause missegregation of the chromosomes during mitosis. Therefore, we examined if other chlamydial infection phenotypes varied between
61 species. We performed a mitotic index to determine if the species of Chlamydia caused mitosis to occur at dif ferent rates. We found that all three species of Chlamydia tested caused a comparable rate of mitosis with no significant difference when comparing the mitotic index ( Figure 4 1C) Similarly, centrosome amplification did not vary between Chlamydia specie s ( Figure 4 1D) Finally, we observed abnormal spindles, or cell s with greater that two spindle poles, to investigate declustered centrosomes. We found a dramatic difference in the species ability to decluster supernumerary centrosomes. While L2 cause g reater than two spindle poles in 70.1 2.6% of mitotic cells, GPIC and MoPn only caused greater than t wo spindle poles in 31.8 1.8% and 51.0 2.0% of mitotic cells, respectively ( Figure 4 1E) We hypothesize that the difference in centrosome decluster ing accounts for the varied multinucleation because the rate of abnormal spindles mimicked the rate of multinucleation. Treating Cells with Griseofulvin Increases Multinucleation Rate We hypothesized that if we induced centrosome declus tering in GPIC infe cted cells, we would observe the same rate of multinucleation as compared to L2infected centrosomes. GPIC induced multinucleation increased from 10.9 0.9 % when untreated to 42.6 1.4% when treated with griseofulvin. This multinucleation rate was not a significant difference compared to 49.2 6.4% for L2infected and griseofulvin treated cells ( Figure 4 2B) Next, we compared abnormal spindles in infected and griseoful vin treated cells to ensure that this change in multinucleation was indeed due to declustering of centrosomes. Uninfected cells treated with griseofulvin had greater than two spindles poles in 52 3% of mitotic cells. When cells were infected with GPIC and treated with
62 griseofulvin this rate increased to 77 10%. Cells infected with L2 and treated with griseofulvin had a multipolarity rate of 77 5 % which was not signific ant compared to the GPIC group ( Figure 4 2C) The mitotic index was calculated from these same cells to ensure that a change in mitotic index for the two species had not occurred. The mitotic ind ex for uninfected, griseofulvin treated cells was 26.2 0.6% that decreased to 16.8 1.4% for GPIC infected cells and 19.5 5.3% f or L2 i nfected cells ( Figure 4 2D) There was no significant difference between the species and the lowered mitotic index indicates that both species of Chlamydia can override the SAC. The SAC should be activated in griseofulvin treated cells due to declustered centrosomes. We conclude that the varying rates of multinucleation are due to the species ability to decluster centrosomes. Centrosomes Are Mislocalized and Declustered during Interphase Centrosomes are normally located in a perinuclear position during interphase. We hypothesized that abnormal spindles would correlate to mispositioned centrosomes in the cell during interphase. We m easured the centrosome to nucleus distance in HeLa cells infected with the different species of Chlamydia to test this hypot hesis. As expected, 79% of the ce of the nucleus. When infected with GPIC or MoPn the centrosomes were pulled away from the nucleus, but 75% and 81% of GPIC and MoPninfected cells positioned Conversely, when the cells were infected with L2, only 55% of the centrosomes were within 5 was a long draw n nucleus [Figure 4 3B]. The centrosome to nucleus distance between species was
63 significantly different by ANOVA. This suggests that the inclusions f ormed by the chlamydial species interact differently with the centrosome. Previously we demonstrated that C. trachomatis L2 infection causes a declustering of centrosomes during interphase in neuroblastoma cells (Knowlton et al., 2011). Neuroblastoma cells have around 810 centrosomes and are therefore a useful cell line to study declustered cent rosomes. The extent of centrosome declustering is determined by measuring the space occupied within a bounding circle. Uninfected cells cluste red the centrosomes within 148 84 2 (Figure 4 4B) There was not a significant difference compared to uninfected cells in centrosome spread when cells were infected with GPIC or MoPn. The MoPn was 122 71 When neuroblastoma cells were infected with L2 the cent rosome spread increased to 229 189 There was a long tail of extremely spread out centrosomes, similar to what was observed in the centrosome to nuclei distance. The centrosome spread increase was significant with a p value of <0.002. Declustered Centrosomes Cause a Disorganized Mitosis All species of Chlamydia drive cell progression through mitosis more rapidly than uninfected cells, but vary in their ability to decluster centrosomes. Therefore, cells infected with L2 should undergo a more disorganized mitosis compared to the other species of Chlamydia We categorized the mitotic cells in each population to deter mine if the species of Chlamydia altered the phases of mitosis differently. In uninfected cells, 11.0 3.2% were in prophase, 22.8 1.3% in prometaphase, 36.3 5.0% in metaphase, 14.6 1.2% in anaphase, and 15.3 4.1% in telophase. The phases of mit osis did not change when cells were infected with GPIC. In GPIC infected cells, 5.6 2.3% were in prophase, 28.7 1.9% in prometaphase, 38.8 2.8% in metaphase,
64 16.7 3.1% in anaphase, and 10.1 1.1% in telophase. However, when cells were infected wi th MoPn or L2, there was a change in the phases of mitosis, with a significant shift from meta phase to prometaphase. In MoPn infected cells 8.2 0.1% were in prophase, 43.6 2.5% in prometaphase, 28.4 3.3% in metaphase, 9.6 1.0 % in anaphase, and 10.1 2.2% in telophase. Similarly, in L2infected cells 5.1 1.0% were in prophase, 51.9 1.4% in prometaphase, 26.2 1.9% in metaphase, 8.1 1.2 % in anaphas e, and 8.7 1.4% in telophase ( Figure 4 5A) Due to the dramatic change in the prometaphase to metaphase ratio, we examined only those two phases. Uninfected cells have a prometa phase to metaphase ratio of 0.6 0.1 (Figure 4 5B) GPIC did not exhibit a significant difference with a ratio of 0.7 However there was a dramatic difference in MoP n and L2infected cells compared to uninf ected cells with a ratio of 1.5 0.3 and 2.0 0.1 with p values of <0.05 and <0.001, respectively. The difference between prometaphase and metaphase is that in metaphase, chromosomes are aligned at the metaphase plate. This shift suggests that MoPnand L2 infected cells require more time to organize their chromosomes, but because of the shortened mitosis, some cells go directly from prometaphase to anaphase. If cells exit mitosis before chromosomes are aligned then we should observe more DNA segregation errors. To test this hypothesis, we scored cells exiting mitosis for DNA bridges. In uninfected cells DNA bridges were present in 6.4 1.1 % of cells that exited mitosis (Figure 4 6B) This increased to 13.5 1.6 % in GPIC infected cells, 24.8 2.5 % in MoPn infected cells and 29.7 3.8% in L2infected cells. MoPn and L2 infected populations demonstrated a significant increase with a pvalue >0.005. These
65 numbers suggest that centrosome declustering is necessary for DNA bridges in infected cells. Discussion We have previously shown that C. trachomatis L2 causes multinucleation through DNA bridging causing cytokinesis failure (Brown et al., 2012). In this study we demonstrated multinucleation of the host cell and declustered centrosomes are not conserved among Chlamydia spp ., but all species override the SAC and amplify centrosomes. To determine if declustered centrosomes correlated to increased multinucleation rate, we infected cells with GPIC and treate d the cells with the centrosomedeclustering agent griseofulvin. Host cells infected with GPIC and treated with griseofulvin demonstrated a similar rate of multipolar mitosis and multinucleation as C. trachomatis infected cells. These findings suggest that dec lustered centrosomes are required for the high rates of multinucleation in host cells. Normally, cells with amplified centrosomes have the innate ability to cluster supernumerary centrosomes to suppress multipolar spindles (Ring et al., 1982). Since C. tr achomatis amplified centrosomes but did not cluster centrosomes, infection must disrupt the clustering process (Quintyne et al., 2005; Saunders, 2005). The host cell demonstrates defects in centrosome clustering beginning in interphase, long before the mi totic spindle has formed. In our studies, we observed that centrosomes were pulled away from the nucleus and spread apart due to C. trachomatis infection. Declustered centrosomes would require correction before mitosis for a normal cell division. The c ells ability to maintain clustered supernumerary centrosomes is an active process that requires both an intact SAC and proper organization of the spindles by microtubule motor proteins (Kwon et al., 2008). C. trachomatis infection overrides the
66 SAC but d oes not affect nuclear mitotic apparatus protein (NuMa) or dynein trafficking (Knowlton et al., 2011; Brown et al., 2012). In this study we demonstrated that all three chlamydial species studied caused a decreased mitotic index, indicating a shortened mito sis and overriding the SAC. We hypothesize that in Chlamydia infected cells, the inclusions interaction with the centrosomes may cause the variation observed in declustering. De clustered centrosomes promote a disorganized mitosis. Therefore we hypothesized that declustered centrosomes would correlate to a shift from metaphase to prometaphase in infected cells. In this study we demonstrated that C. trachomatis had the largest metaphase to prometaphase shift that correlated to declustered centrosomes. Finally, we investigated variation in DNA tangles due to infection with different species. C. trachomatis causes DNA in the midbody of mitotic host cells that results in cytokinesis failure (Brown et al., 2012). In this study we observed that DNA in the mi dbody is correlated to declustered centrosomes. C. caviae infected cells decluster centrosomes less than C. trachomatis infected cells, which results in a lower rate of multinucleation. Declustered centrosomes promote multipolar and pseudobipolar mitosi s, resulting in chromosomal segregation errors. The presence of chromosomal segregation errors cause the cell to fail in cytokinesis in order protect the integrity of the DNA through the NoCut pathway (Norden et al., 2006; Steigemann and Gerlich, 2009a). W e therefore propose a new model in which all species of Chlamydia override the SAC and amplify centrosomes, but the studied species cause varied degrees of declustered centrosomes that correlate to the rate of multinucleation ( Figure 4 7 ).
67 Therefore during a C. trachomatis infection, host cells enter mitosis with declustered centrosomes. Before the cell has time to correct the declustered centrosomes, Chlamydia bypasses the SAC. This results in lagging chromosomes and cytokinesis failure. Conversely, GPIC and MoPn decluster centrosomes less, so when the SAC is bypassed there are fewer lagging chromosomes and cytokinesis is completed. Previous C. trachomatis infections have been epidemiologically linked to cervical cancer. In this study we have elucidated a mechanism by which Chlamydia could contribute to cellular transformation. Infection with C. trachomatis may contribute to cellular transformation and cervical cancer, by inducing aneuploidy through DNA segregation errors and cytokinesis failure
68 Figure 4 1. Multinucleation with different species A) HeLa cells were infected with Chlamydia trachomatis (L2), C. muridarum (MoPn) or C. caviae (GPIC) for 40 hours. Cells were stained with anti tubulin (green), human s erum (red), and Draq5 (blue). B) U ninfected cells had a multinucleation rate of 2.9 0.6%, which increased to 64.7 2.5% in L2 infected cells. GPIC and MoPn also demonstrated an increase multinucleation rate of 10.9 0.9 % and 28.1 0.6%. Compared to uninfected, GPIC, MoPn, and L2 i nfected cells were significantly different (ANOVA) N=3 experiments with at l east 600 cells per experiment. C) Number of mitotic cells compared to total cells was counted for the mitotic index. Uninfected cells had a mitotic index of 7.7 0.5%, while GPIC, MoPn and L2 had a mitotic index of 4.6 0.2%, 5.0 0.3% and 4.5 0.4%, respectively. There was significance difference in means between uninfected GPIC, MoPn, and L2 (ANOVA), but there was not a significant difference between the species. N=3 experiments with at least 1500 cells per experiment. D) Percent of mitotic cells that contained more than 2 spindle poles were evaluated. Uninfected cells had multipolar spindles 6.8 0.9% of the time. When infected with the different species the abnormal s pindle rate was 31.8 1.8% (GPIC), 51.0 2.0% (MoPn) and 70.1 2.6% (L2). There was significance difference of means between uninfected, and all species of Chlamydia (ANOVA), and a significance of p<0.001 between species. N=3 experiments with at l east 1 00 cells per experiment. E) Centrosomes per cell were counted to determine the centrosome amplification in infected cells.
69 Uninfected had 1.8 0.2 centrosomes per cell. The infected cells had 2.3 (GPIC), 2.4 0.1 (MoPn) and 2.7 0.1 (L2) centrosomes per cell. There was significance difference in means of uninfect ed and all species (ANOVA) a significance of p<0.005 of L2 compared to GPIC or MoPn. There was not a significant difference between GPIC and MoPn. N=3 experiments with at least 150 cells per experiment.
70 Figure 4 2. Declustering of centrosomes by griseofulvin. A) HeLa cells were infected with further 16 hours. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). B) Multinucleation of infected cells was evaluated. Uninfected, GPIC and L2 infec ted cells had a percent multinucleation of 27.6 3.6%, 42.6 1.4% and 49.2 6.4%. There was significance of uninfected and GPIC or L2 infected of p<0.002, but no significant difference between species. N=3 experiments with at l east 600 cells per experi ment. C) Percent of mitotic cells that contained more than 2 spindle poles were evaluated. When treated with griseofulvin, uninfected c ells had abnormal spindles 52 3 %. Cells infected with GPIC or L2 had abnormal spindles 77 10% and 77 5%, respectiv ely. There was significance of uninfected and GPIC or L2 infected of p<0.02, but no significant difference between species. N=3 experiments with at l east 100 cells per experiment. D) Mitotic index was performed on griseofulvin treated cells. Uninfected, GPIC and L2 infected cells had a mitotic index of 26.2 0.6%, 16.8 1.4% and 19.5 5.3%. There was significance of uninfected and GPIC or L2 infected of p<0.05, but no significant difference between species. N=3 experiments with at least 1500 cells per ex periment
71 Figure 4 3. Centrosome to nucleus distance. A) HeLa cells were infected with L2, or GPIC for 40 hours. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). B) The distance between the closest centrosome and the nuclei was measured. Distances are presented in a histogram. of the nucleus. GPIC and MoPn infected cells have centrosomes shifted away L2 has a long tail on the There is a significant difference in the mean distance between populations (ANOVA) N=100 cells, per population.
72 Figure 4 4. Centrosome clust ering in neuroblastoma cells. A) Neuroblastoma cells were infected with L2, MoPn or GPIC for 40 hours. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). B) The centrosome spread was measured with a bounding circle and was graphed wi th a box and whisker plot. Uninfected cells clustered the centrosomes within 147.6 83.6 2. When infected with GPIC, MoPn or L2 the centrosome where contained There was not a sigi ficant difference between uninfected and GPIC or MoPn infected cells (ANOVA) but L2 had a significance of p<0.001 compared the other species. N=100 cells.
73 Figure 4 5. Mitosis is disorganized upon infection with L2. HeLa cells were infected with L2, Mo Pn or GPIC for 40 hours. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). A) The stages of cells in mitosis were analyzed. GPIC infected cells have a similar profile to the stages of mitosis of uninfected cells. Both M oPn and L2 show a shift to predominantly prometaphase, though L2 demonstrates are larger shift. N=3 experiments with 200 cells per experiment. B) The prometaphase to metaphase ratio was evaluated. Unin fected cells had a ratio of 0.6 0.1, while GPIC, Mo Pn and L2 had a ratio of 0.7, 1.5 0.3 and 2.0 0.1 respectively. The groups had significantly different means (ANOVA) N=3 experiments with 200 cells per experiment.
74 Figure 46. DNA segregation errors c orrelate with multinucleation. A) HeLa cells were infected with L2, MoPn or GPIC for 40 hours. Cells were stained with anti tubulin (green), human serum (red), and Draq5 (blue). B) Cells exiting mitosis were evaluated for DNA bridges and lagging chromosomes. Uninfected cells had a DNA bridging rate of 6.4 1.1%. Upon infection, cells infected with G PIC had a rate of 13.5 1.6%, MoPn had a rate of 24.8 2.5% and L2 had a rate of 29.7 3.8%. The groups had significantly different means (ANOVA). There was a significance of p> 0.005 for any infecti on compared to uninfected. N=3 experiments with 100 cells per experiment.
75 Figure 47 Model of differences in multinucleation. Upon infection centrosomes are amplified in host cells. In L2 infected cells, centrosomes are pulled away from the nucleus and dispersed throughout the cell, resulting in declustered centrosome. This leads lagging chromosomes, which when not cleared from the midbody, results in the cell aborting cytokinesis. In GPIC infected cells, centrosome positioning is only disturbed sl ightly, allowing the centrosomes to cluster during mitosis. This results in a normal mitosis and completion of cytokinesis.
76 CHAPTER 5 DISCUSSION Hanahan and Weinberg outlined eight hallmarks of cancer that enable tumor growth and two enabling characteristics that allow hallmark acquisition to be possible. (Hanahan and Weinberg, 2000; 2011). Chlamydia infection has been shown to enable two hallmarks in infected host cells: sustaining proliferative signaling and resisting cell death (Jungas et al., 2004; Knowlton et al., 2013). Importantly, Chlamydia also contributes to the two enabling characteristics: tumor promoting inflammation and genome instability. Chlamydial infection promotes a strong inflammatory response that fosters an environment for transf ormation (Roshick et al., 2006; Rusconi and Greub, 2011). Furthermore, Chlamydia can induce disorganized mitosis and multinucleation leading to genomic instability (Knowlton et al., 2011; Sun et al., 2011). Our model suggests that genomic instability due to Chlamydia infection contributes to the higher incidence of cervical cancer in women with a prior C. trachomatis infection. Multinucleation of host cells due to a Chlamydia infection has been recognized for many years and therefore we aimed to determine the mechanism by which host cells become multinucleated (Horoschak and Moulder, 1978; Bose and Liebhaber, 1979; Greene and Zhong, 2003). We demonstrated multinucleation occurs through cytokinesis failure and ruled out a contribution by cell fusion. In our study, we examined the steps in cytokinesis to determine where cytokinesis failure occurs. Two studies have suggested cytokinesis failure occurs because Chlamydia acts as a large bolus of material that causes steric interference, which leads to cytokinesis failure (Campbell et al., 1989; Sun et al., 2011). When we examined the first two steps in cytokinesis we found no change in the cleavage furrow or the percentage of cells that formed midbodies. Furthermore,
77 multinucleation was not induced by Coxiella burnetii another obligate intracellular bacteria that reside in a large vacuole within the cell. This suggested the failure in cytokinesis was not due to steric interference and multinucleation of the host cell was chlamydial specific. Since steric int erference did not account for a large portion of failures in cytokinesis we examined the fi nal step of cytokinesis, abscission. We demonstrated that cells fail extremely late in cytokinesis Using live cell imaging, we observed differential expression of GFP bicaudal D1 between the forming daughter cells, suggesting an inhibition of diffusion. This only occurs once the midbody has formed, so therefore cytokinesis fails at abscission. Since the most likely cause of abscission failure is DNA segregation er rors, we examined the effect chlamydial infection had on mitosis. We found C. trachomatis infected cells underwent mitosis more rapidly and were able to escape a monastrol block, which suggested chlamydial infection had weakened the SAC. We found that a rapid, disorganized mitosis led to chromosome segregation errors and confirmed that the population of cells that failed cytokinesis was the same population that contained DNA in the midbody of the cell. This evidence leads us to our model of C. t r achomati sinduced DNA segregation errors during mitosis are the leading cause of cytokinesis failure in infected cells. We proposed a model in which Chlamydia s ability to override the SAC was due to degradation of cyclin B1 and securin by the protease CPAF. Rec ently published data demonstrated that degradation observed in western blot analysis was due to enhanced CPAF activity in the sample buffer. When proteins were collected in 8M urea, cyclin B1 was not degraded during the course of the Chlamydia infection ( Chen et al., 2012). We confirmed these findings by western blot analysis and found cyclin B1 and securin were
78 not cleaved in detectable levels when lysed in 8M urea (data not shown). We plan to examine CPAF further as the cause of premature mitotic exit because cyclin B1 and securin are not degraded when a CPAF inhibitor is added to the sample buffer, suggesting under certain conditions CPAF can degrade cyclin B1 and securin (Brown et al., 2012). Furthermore, both the cell cycle and developmental cycle ar e asynchronous, which complicates western blot analysis. Therefore, we propose that a chlamydial protease, that may or may not be CPAF, acts on a different target in the Spindle Assembly Checkpoint pathway. We will further investigate the way Chlamydia o verrides the SAC with live cell imaging to study the events of a single cell at a time. Next, we aimed to determine if multinucleation was conserved among divergent Chlamydia species. C. trachomatis causes amplified centrosomes, declustered centrosomes, premature mitotic exit and multinucleation (Grieshaber et al., 2006; Johnson et al., 2009; Knowlton et al., 2011; Brown et al., 2012). In order to investigate which phenotypes were conserved among species, we compared cytological change in three species of Chlamydia. All three species investigated caused shorten mitosis and centrosome amplification, demonstrating that these phenot ypes are highly conserved among Chlamydia We found that multinucleation rates and multipolar spindle rates varied among species. Both phenotypes followed the same trend, suggesting that multipolar spindles were the cause of the multinucleation. To test this theory, we forced centrosomes to decluster during infection using griseofulvin. From this, we conclude that shortened mitosi s, amplified and declustered centrosomes are all necessary for cytokinesis failure.
79 We have shown that Chlamydia overrides the SAC, which forces the cell to undergo mitosis more rapidly. Mislocalized centrosomes require extra time to form a pseudobipola r spindle, which is controlled by the SAC. We found that centrosomes remained near the nucleus and clustered when host cells were infected with GPIC and MoPn, but centrosomes moved away from the nucleus and spread throughout the cell when infected with L2. Declustered centrosomes cause a disorganized mitosis in several ways. The most striking phenotype is multipolar mitosis, but some only undergo a transitory multipolar spindle intermediate before extra centrosomes are clustered into a pseudobipolar spi ndle (Ganem et al., 2009). This transitory state promotes merotelic attachments, causing chromosome segregation errors. In the future we plan to determine how the chlamydial inclusions from the three species interact with the centrosomes. The differences in inclusion centrosome interaction would account for the variation in centrosome spread during interphase and the rate of multipolar mitosis. The chlamydial inclusion interacts with the centrosome through dynein, but the specific chlamydial proteins have not been identified (Grieshaber et al., 2003). We plan to identify the chlamydial proteins in the inclusion that interact with the centrosome. Species variation in this unknown protein could account for the differences in centrosome spread. Next, we plan to determine if specific chromosomes become trapped in the midbody of infected cells, or if trapped chromosomes are random. We will synchronized the cells with a double thymidine block and then perform fluorescence in situ hybridization (FISH) on cells ex iting mitosis. This experiment will provide new insites the mechanism driving chromosome segregation errors in Chlamydiainfected cells.
80 Finally our lab will continue studying the mechanism by which chlamydial infection causes premature exit from mitos is and centrosome amplification. Because these two phenotypes are conserved among all species, we hypothesize that one chlamydial effector causes both these phenotypes. The protein complex termed cohesin prevents mitotic exit and centrosome replication. C ohesin forms a ring structure around sister chromosomes and centrosomes, inhibiting sister chromosome and centrosome disengagement (Wittmann et al., 2001; Schckel et al., 2011). In uninfected cells the protease separase cleaves cohesin on both chromosom es and centrosomes during mitotic exit. We hypothesize that a chlamydial protease cleaves cohesin prematurely. Preliminary data from our lab suggests that the targeted protein is Ssc1, part of the ring structure of the cohesin complex. Chlamydial infec tion causes centrosome amplification during G2 and in this study we demonstrated that Chlamydia infected cells spend more time in G2 than uninfected cells (Johnson et al., 2009). We hypothesize that the elongated G2 phase, paired with a chlamydial protease working as a separase mimic, allows centrioles to prematurely separate and reduplicate. We will explore this hypothesis using live cell imaging and a separase sensor construct provided by Dr. Stemmann (Schckel et al., 2011). The construct consists of a fusion of four functional domains: a targeting domain, the mCherry fluorescent protein, the Ssc1 cleavage domain, followed by the EGFP protein. The provided construct has H2B as the targeting domain to cause the fusion protein to locate to the chromosomes We will change this targeting domain to centrin, in order to also visualize centrosome dynamics. Live cell imaging with these constructs will
81 provide us with further insight into the mechanism of cytokinesis failure. More studies need to be performed to fully understand host cell cytokinesis failure; however, the results presented here provide new insights into Chlamydia s role in multinucleation, centrosome declustering and genomic instability.
82 LIST OF REFERENCES Alibek, K., Karatayeva, N., and B ekniyazov, I. (2012). The role of infectious agents in urogenital cancers. Infect. Agents Cancer 7 35. Alzhanov, D.T., Weeks, S.K., Burnett, J.R., and Rockey, D.D. (2009). Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223. BMC Microbiol. 9, 2. Anderhub, S.J., Krmer, A., and Maier, B. (2012). Centrosome amplification in tumorigenesis. Cancer Lett. 322 8 17. Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., and Mann, M. (2003). Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426 570 574. Andreassen, P.R., Lohez, O.D., Lacroix, F.B., and Margolis, R.L. (2001). Tetraploid state induces p53dependent arrest of nontransformed mammalian cells in G1. Mol B iol Cell 12, 1315 1328. Anttila, T., Saikku, P., Koskela, P., Bloigu, A., Dillner, J., Ikheimo, I., Jellum, E., Lehtinen, M., Lenner, P., Hakulinen, T., et al. (2001). Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell c arcinoma. Jama 285 47 51. Baker, D.J., Dawlaty, M.M., Galardy, P., and van Deursen, J.M. (2007). Mitotic regulation of the anaphasepromoting complex. Cell Mol Life Sci 64 589 600. Balsara, Z.R., Misaghi, S., Lafave, J.N., and Starnbach, M.N. (2006). Chl amydia trachomatis infection induces cleavage of the mitotic cyclin B1. Infection and Immunity 74, 5602 5608. Belland, R., Ojcius, D.M., and Byrne, G.I. (2004). Chlamydia. Nat Rev Microbiol 2 530 531. Benanti, J.A. (2012). Coordination of cell growth and division by the ubiquitinproteasome system. Seminars in Cell and Developmental Biology 23, 492 498. Bose, S.K., and Liebhaber, H. (1979). Deoxyribonucleic acid synthesis, cell cycle progression, and division of Chlamydiainfected HeLa 229 cells. Infection and Immunity 24, 953 957. Boveri, T. (2008). Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Harris. J Cell Sci 121 Suppl 1 1 84. Brinkley, B.R. (2001). Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol 11, 18 21.
83 Brinkley, B.R., and Goepfert, T.M. (1998). Supernumerary centrosomes and cancer: Boveri's hypothesis resurrected. Cell Motil. Cytoskeleton 41, 281 288. Brown, H.M., Knowlton, A.E., and Grieshab er, S.S. (2012). Chlamydial Infection Induces Host Cytokinesis Failure at Abscission. Cell Microbiol. Burton, M.J., and Mabey, D.C.W. (2009). The global burden of trachoma: a review. PLoS Negl Trop Dis 3 e460. Caballe, A., and Martin Serrano, J. (2011). E SCRT machinery and cytokinesis: the road to daughter cell separation. Traffic 12, 1318 1326. Campbell, S., Richmond, S.J., and Yates, P. (1989). The development of Chlamydia trachomatis inclusions within the host eukaryotic cell during interphase and mitosis. J. Gen. Microbiol. 135 1153 1165. Cates, W., and Wasserheit, J.N. (1991). Genital chlamydial infections: epidemiology and reproductive sequelae. Am. J. Obstet. Gynecol. 164 1771 1781. Chan, J.Y. (2011). A clinical overview of centrosome amplification in human cancers. Int. J. Biol. Sci. 7 1122 1144. Chen, A.L., Johnson, K.A., Lee, J.K., Stterlin, C., and Tan, M. (2012). CPAF: a Chlamydial protease in search of an authentic substrate. PLoS Pathog 8 e1002842. Cimini, D., Howell, B., Maddox, P., Khod jakov, A., Degrassi, F., and Salmon, E.D. (2001). Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J Cell Biol 153, 517 527. Clmenson, C., and Marsolier Kergoat, M.C. (2009). ScienceDirect.com DNA Repair DNA damage checkpoint inactivation: Adaptation and recovery. DNA Repair. Clifton, D.R., Dooley, C.A., Grieshaber, S.S., Carabeo, R.A., Fields, K.A., and Hackstadt, T. (2005). Tyrosine phosphorylation of the chlamydial effector protein Tarp is spec ies specific and not required for recruitment of actin. Infection and Immunity 73, 3860 3868. Clifton, D.R., Fields, K.A., Grieshaber, S.S., Dooley, C.A., Fischer, E.R., Mead, D.J., Carabeo, R.A., and Hackstadt, T. (2004). A chlamydial type III translocat ed protein is tyrosinephosphorylated at the site of entry and associated with recruitment of actin. Proc Natl Acad Sci USA 101 10166 10171. de Martel, C., Ferlay, J., Franceschi, S., Vignat, J., Bray, F., Forman, D., and Plummer, M. (2012). Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol. 13 607 615.
84 Dehoux, P., Flores, R., Dauga, C., Zhong, G., and Subtil, A. (2011). Multi genome identification and characterization of chlamydiaespecific type I II secretion substrates: the Inc proteins. BMC Genomics 12 109. Dong, F., Pirbhai, M., Zhong, Y., and Zhong, G. (2004). Cleavagedependent activation of a chlamydiasecreted protease. Mol Microbiol 52, 1487 1494. Duelli, D., and Lazebnik, Y. (2007) Cell t o cell fusion as a link between viruses and cancer. Nat Rev Cancer. 7, 968976. Eggert, U.S., Mitchison, T.J., and Field, C.M. (2006). Animal cytokinesis: from parts list to mechanisms. Annu. Rev. Biochem. 75 543 566. Ferguson, R.L., and Maller, J.L. (201 0). Centrosomal localization of cyclin E Cdk2 is required for initiation of DNA synthesis. Curr Biol 20, 856 860. Fields, K.A., and Hackstadt, T. (2002). The chlamydial inclusion: escape from the endocytic pathway. Annu Rev Cell Dev Biol 18, 221 245. Friis R.R. (1972). Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol 110, 706 721. Fujiwara, T., Bandi, M., Nitta, M., Ivanova, E.V., Bronson, R.T., and Pellman, D. (2005). Cytokinesis failu re generating tetraploids promotes tumorigenesis in p53null cells. Nature 437 1043 1047. Fukasawa, K. (2007). Oncogenes and tumour suppressors take on centrosomes. Nat Rev Cancer 7 911 924. Ganem, N.J., and Pellman, D. (2007). Limiting the proliferation of polyploid cells. Cell 131, 437 440. Ganem, N.J., and Pellman, D. (2012). Linking abnormal mitosis to the acquisition of DNA damage. J Cell Biol 199 871 881. Ganem, N.J., Godinho, S.A., and Pellman, D. (2009). A mechanism linking extra centrosomes to c hromosomal instability. Nature 460 278 282. Gavet, O., and Pines, J. (2010). Progressive activation of CyclinB1Cdk1 coordinates entry to mitosis. Dev Cell 18, 533 543. Glotzer, M. (2001). Animal cell cytokinesis. Annu Rev Cell Dev Biol 17, 351 386. Glotz er, M. (2005). The Molecular Requirements for Cytokinesis. Science 307, 1735 1739. Gordon, D.J., Resio, B., and Pellman, D. (2012). Causes and consequences of aneuploidy in cancer. Nat. Rev. Genet. 13, 189 203.
85 Gould, R.R., and Borisy, G.G. (1977). The pe ricentriolar material in Chinese hamster ovary cells nucleates microtubule formation. J Cell Biol 73 601 615. Greene, W., and Zhong, G. (2003). Inhibition of host cell cytokinesis by Chlamydia trachomatis infection. J Infect 47 45 51. Grieshaber, S.S., Grieshaber, N.A., and Hackstadt, T. (2003). Chlamydia trachomatis uses host cell dynein to traffic to the microtubuleorganizing center in a p50 dynamitinindependent process. J Cell Sci 116 3793 3802. Grieshaber, S.S., Grieshaber, N.A., Miller, N., and Ha ckstadt, T. (2006). Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities. Traffic 7 940 949. Hackstadt, T., Rockey, D.D., Heinzen, R.A., and Scidmore, M.A. (1996). Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. Embo J 15, 964 977. Hackstadt, T., Scidmore, M.A., and Rockey, D.D. (1995). Lipid metabolism in Chlamydia trachomatis infected cells: di rected trafficking of Golgi derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci USA 92, 4877 4881. Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57 70. Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of canc er: the next generation. Cell 144, 646 674. Hartwell, L.H., and Weinert, T.A. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science 246 629 634. Heinzen, R.A., Hackstadt, T., Samuel, J.E. (1999) Developmental biology of Coxiella burnettii Trends in Microbiology 7, 149 154. Heinzen, R.A., Scidmore, M.A., Rockey, D.D., Hackstadt, T. (1996) Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachom atis Infect. Immun. 64, 796 809. Horoschak, K.D., and Moulder, J.W. (1978). Division of single host cells after infection with chlamydiae. Infection and Immunity 19 281 286. Hu, V.H., Harding Esch, E.M., Burton, M.J., Bailey, R.L., Kadimpeul, J., and Ma bey, D.C.W. (2010). Epidemiology and control of trachoma: systematic review. Trop Med Int Health 15, 673691. Huang, Z., Feng, Y., Chen, D., Wu, X., Huang, S., Wang, X., Xiao, X., Li, W., Huang, N., Gu, L., et al. (2008). Structural basis for activation and inhibition of the secreted chlamydia protease CPAF. Cell Host Microbe 4, 529 542.
8 6 Iliffe Lee, E.R., and McClarty, G. (2000). Regulation of carbon metabolism in Chlamydia trachomatis. Mol Microbiol 38 20 30. Jirawatnotai, S., Hu, Y., Livingston, D.M., and Sicinski, P. (2012). Proteomic identification of a direct role for cyclin d1 in DNA damage repair. Cancer Res. 72, 4289 4293. Johnson, K.A., Tan, M., and Stterlin, C. (2009). Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway. Cell Microbiol 11, 1064 1073. Jorgensen, I., Bednar, M.M., Amin, V., Davis, B.K., Ting, J.P.Y., McCafferty, D.G., and Valdivia, R.H. (2011). The Chlamydia Protease CPAF Regulates Host and Bacterial Prote ins to Maintain Pathogen Vacuole Integrity and Promote Virulence. Cell Host Microbe 10, 21 32. Jungas, T., Verbeke, P., Darville, T., and Ojcius, D.M. (2004). Cell death, BAX activation, and HMGB1 release during infection with Chlamydia. Microbes and Infec tion 6 1145 1155. Kapoor, T.M., Mitchison, T.J. (2001) Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix. J Cell Biol 154, 1125 1133. Kim, S., Yu, H. (2011). Mutual regulation between the spindle checkpoint and APC/C. Seminars in Cell and Developmental Biology 22, 551558. Kevin Hybiske, R.S.S. (2007) Mechanisms of host cell exit by the intraceullular bacterium Chlamydia. Proc Natl Acad Sci U.S.A 104, 1143011435. King R.W. (2008) When 2+2=5: the origins and fates of aneuploid and tetraploid cells. Biochim. Biophys. Acta 1786, 4 14. Kiviat, N.B., Paavonen, J.A., Brockway, J., Critchlow, C.W., Brunham, R.C., Stevens, C.E., Stamm, W.E., Kuo, C.C., DeRouen, T., and Holmes, K.K. (1985). Cytologic manifestations of cervical and vaginal infections. I. Epithelial and inflammatory cellular changes. JAMA : The Journal Of The American Medical Association 253, 98996. Knowlton, A.E., Brown, H.M., Richards, T.S., Andreolas, L.A., Patel, R.K., and Grieshaber, S.S. (2011). C hlamydia trachomatis infection causes mitotic spindle pole defects independently from its effects on centrosome amplification. Traffic. Knowlton, A.E., Fowler, L.J., Patel, R.K., Wallet, S.M., and Grieshaber, S.S. (2013). Chlamydia Induces Anchorage Independence in 3T3 Cells and Detrimental Cytological Defects in an Infection Model. PLoS ONE 8 e54022.
87 Korzeniewski, N., Spardy, N., Duensing, A., Duensing, S. (2011) Genomic instability and cancer: lessons learned from human papillomaviruses. Cancer Lett. 305 113 122. Koskela, P., Anttila, T., Bjrge, T., Brunsvig, A., Dillner, J., Hakama, M., Hakulinen, T., Jellum, E., Lehtinen, M., Lenner, P., et al. (2000). Chlamydia trachomatis infection as a risk factor for invasive cervical cancer. Int J Cancer 85 35 39. Kuriyama, R., and Borisy, G.G. (1981). Centriole cycle in Chinese hamster ovary cells as determined by wholemount electron microscopy. J Cell Biol 91 814 821. Kwon, M., Godinho, S.A., Chandhok, N.S., Ganem, N.J., Azioune, A., Thery, M., and Pellman, D. (2008). Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev 22, 2189 2203. Liu, B., Hong, S., Tang, Z., Yu, H., Giam, C.Z., (2005) HTLV I Tax directly binds the Cdc20associated anaphasepromoting complex and ac tivates it ahead of schedule. Proc. Natl. Acad. Sci. U.S.A. 102, 63 68. Lohez, O.D., Reynaud, C., Borel, R., Andreassen P.R., Marglois, R.L. (2003) Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53. J Cell BIol 161, 6777. Madeleine, M.M., Anttila, T., Schwartz, S.M., Saikku, P., Leinonen, M., Carter, J.J., Wurscher, M., Johnson, L.G., Galloway, D.A., and Daling, J.R. (2007). Risk of cervical cancer associated with Chlamydia trachomatis antibodies by histology, HPV type and HPV cofactors. Int J Cancer 120 650 655. Marangos, P., and Carroll, J. (2008). Securin regulates entry into M phase by modulating the stability of cyclin B. Nat Cell Biol 10, 445 451. Matanis, T., Akhmanova, A., Wulf, P., Del Nery, E., Weide, T., Stepanova, T., Galjart, N., et al. (2002) Bicaudal D regulates COPI independent Golgi ER transport by recruiting the dyneindynactin motor complex. Nat. Cell Biol. 4, 986 992. Matsumoto, K., Yasugi, T., Oki, A., Hoshiai, H., Taketani, Y., Kawana, T., and Yoshikawa, H. (2003). Are smoking and chlamydial infection risk factors for CIN? Different results after adjustment for HPV DNA and antibodies. Br. J. Cancer 89, 831 833. McLaughlinDrubin, M.E., Mnger, K. (2009) The human papillomavirus E7 oncoprotein. Virology 384: 335 344. McLean, J.R., Chaix, D., Ohi, M.D., Gould, K.L. (2011) State of the APC/C: organization, function, and structure. Crit. Rev. Biochem. Mol. Biol. 46, 118 136.
88 Mendoza, M., Norden, C., Dur rer, K., Rauter, H., Uhlmann, F., and Barral, Y. (2009). A mechanism for chromosome segregation sensing by the NoCut checkpoint. Nat Cell Biol 11, 477 483. Menoyo, S., Ricco, N., Bru, S., Hernndez Ortega, S., Escot, X., Aldea, M., and Clotet, J. (2013). Phosphateactivated cyclin dependent kinase stabilizes g1 cyclin to trigger cell cycle entry. Mol. Cell. Biol. 33 1273 1284. Moorhead, A.R., Rzomp, K.A., and Scidmore, M.A. (2007). The Rab6 effector Bicaudal D1 associates with Chlamydia trachomatis inclus ions in a biovar specific manner. Infection and Immunity 75, 781 791. Moulder, J.W. (1991). Interaction of chlamydiae and host cells in vitro. Microbiol Rev 55, 143 190. Nigg, E.A. (2002). Centrosome aberrations: cause or consequence of cancer progression? Nat Rev Cancer 2 815 825. Norden, C., Mendoza, M., Dobbelaere, J., Kotwaliwale, C.V., Biggins, S., and Barral, Y. (2006). The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage. Cell 125 85 98. Norm and, G., King, R.W. (2010) Understanding cytokinesis failure. Adv Exp Med Biol 676, 2755. Paschen, S.A., Christian, J.G., Vier, J., Schmidt, F., Walch, A., Ojcius, D.M., and Hcker, G. (2008). Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease. J Cell Biol 182 117 127. Peters, J.M. (2006). The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 7 644 656. Piel, M., Meyer, P., Khodjakov, A., Rieder, C.L., and Bornens, M. (2000). The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J Cell Biol 149 317 330. Pihan, G.A., Purohit, A., Wallace, J., Knecht, H., Woda, B., Quesenberry, P., and Doxsey, S.J. (19 98). Centrosome defects and genetic instability in malignant tumors. Cancer Res. 58, 3974 3985. Pines, J. (2011). Cubism and the cell cycle: the many faces of the APC/C. Nat Rev Mol Cell Biol. Pisani, P., Parkin, D.M., Muoz, N., and Ferlay, J. (1997). Cancer and infection: estimates of the attributable fraction in 1990. Cancer Epidemiol Biomarkers Prev 6 387 400.
89 Quintyne, N.J., Reing, J.E., Hoffelder, D.R., Gollin, S.M., and Saunders, W.S. (2005). Spindle multipolarity is prevented by centrosomal clustering. Science 307 127 129. Ramos, J.G., Varayoud, J., Bosquiazzo, V.L., Luque, E.H., Muoz deToro, M. (2002) Cellular turnover in the rat uterine cervix and its relationship to estrogen and progesterone receptor dynamics. Biol Reprod 67, 735 742. Read, T.D., Brunham, R.C., Shen, C., Gill, S.R., Heidelberg, J.F., White, O., Hickey, E.K., Peterson, J., Utterback, T., Berry, K., et al. (2000). Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 28, 1397 1406. Rea d, T.D., Myers, G.S.A., Brunham, R.C., Nelson, W.C., Paulsen, I.T., Heidelberg, J., Holtzapple, E., Khouri, H., Federova, N.B., Carty, H.A., et al. (2003). Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of nichespeci fic genes in the evolution of the Chlamydiaceae. Nucleic Acids Res. 31 2134 2147. Ring, D., Hubble, R., and Kirschner, M. (1982). Mitosis in a cell with multiple centrioles. J Cell Biol 94 549 556. Roset Bahmanyar, E., Paavonen, J., Naud, P., Salmern, J ., Chow, S.N., Apter, D., Kitchener, H., Castellsagu, X., Teixeira, J.C., Skinner, S.R., et al. (2012). Prevalence and risk factors for cervical HPV infection and abnormalities in young adult women at enrolment in the multinational PATRICIA trial. Gynecol. Oncol. 127 440 450. Roshick, C., Wood, H., Caldwell, H.D., and McClarty, G. (2006). Comparison of gamma interferonmediated antichlamydial defense mechanisms in human and mouse cells. Infection and Immunity 74, 225 238. Rusconi, B., and Greub, G. (2011). Chlamydiales and the innate immune response: friend or foe? FEMS Immunol Med Microbiol 61, 231 244. Saka, H.A., and Valdivia, R.H. (2010). Acquisition of nutrients by Chlamydiae: unique challenges of living in an intracellular compartment. Curr Opin Mic robiol 13 4 10. SakaueSawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H. Osawa, H., et al. (2008) Visualizing spatiotemporal dynamics of multicellular cell cycle progression. Cell 132, 487 498. Samaras, V., Rafailidis, P.I., Mourtzoukou, E.G., Peppas, G., and Falagas, M.E. (2010). Chronic bacterial and parasitic infections and cancer: a review. J Infect Dev Ctries 4, 267 281 15. Saunders, W. (2005). Centrosomal amplification and spindle multipolarity in cancer cells. Semin. Cancer Biol. 15, 25 32. Schmidt, K., and Nichols, B.J. (2004). A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells. Curr Biol 14 1002 1006.
90 Schckel, L., Mckel, M., Mayer, B., Boos, D., and Stemmann, O. (2011). Cleavage of cohesin rings coordi nates the separation of centrioles and chromatids. Nat Cell Biol 13 966 972. Shaw, A.C., Vandahl, B.B., Larsen, M.R., Roepstorff, P., Gevaert, K., Vandekerckhove, J., Christiansen, G., and Birkelund, S. (2002). Characterization of a secreted Chlamydia pro tease. Cell Microbiol 4 411 424. Smith, J.S., Bosetti, C., Muoz, N., Herrero, R., Bosch, F.X., Eluf Neto, J., Meijer, C.J.L.M., Van Den Brule, A.J.C., Franceschi, S., Peeling, R.W., et al. (2004). Chlamydia trachomatis and invasive cervical cancer: a pooled analysis of the IARC multicentric case control study. Int J Cancer 111 431 439. Smith, J.S., Muoz, N., Herrero, R., Eluf Neto, J., Ngelangel, C., Franceschi, S., Bosch, F.X., Walboomers, J.M.M., and Peeling, R.W. (2002). Evidence for Chlamydia tracho matis as a human papillomavirus cofactor in the etiology of invasive cervical cancer in Brazil and the Philippines. J. Infect. Dis. 185 324 331. Steigemann, P., and Gerlich, D.W. (2009a). An evolutionary conserved checkpoint controls abscission timing. Cell Cycle 8 1814 1815. Steigemann, P., and Gerlich, D.W. (2009b). Cytokinetic abscission: cellular dynamics at the midbody. Trends Cell Biol 19 606 616. Steigemann, P., Wurzenberger, C., Schmitz, M.H.A., Held, M., Guizetti, J., Maar, S., and Gerlich, D.W. (2009). Aurora B mediated abscission checkpoint protects against tetraploidization. Cell 136, 473 484. Stephens, R.S., Kalman, S., Lammel, C., Fan, J., Marathe, R., Aravind, L., Mitchell, W., Olinger, L., Tatusov, R.L., Zhao, Q., et al. (1998). Genome se quence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282 754 759. Sun, H.S., Wilde, A., and Harrison, R.E. (2011). Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in hos t cells. Mol. Cell. Biol. 31 5011 5022. Swanson, J., Eschenbach, D.A., Alexander, E.R., and Holmes, K.K. (1975). Light and electron microscopic study of Chlamydia trachomatis infection of the uterine cervix. J. Infect. Dis. 131 678 687. Todd, W.J., Caldw ell, H.D. (1985). The interaction of Chlamydia trachomatis with host cells: ultrastructural studies of the mechanism of release of a biovar II strain from HeLa 229 cells. J Infect Dis 151, 10371044. Thompson, S.L., Bakhoum, S.F., Compton, D.A. (2010). Mechanisms of chromosomal instability. Curr Biol 20, 28595.
91 Uetake, Y., and Sluder, G. (2004). Cell cycle progression after cleavage failure: mammalian somatic cells do not possess a "tetraploidy checkpoint". J Cell Biol 165 609 615. Uhlmann, F., Lottspei ch, F., and Nasmyth, K. (1999). Sister chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400 37 42. van Leuken, R., Clijsters, L., and Wolthuis, R. (2008). To cell cycle, swing the APC/C. Biochim Biophys Ac ta 1786, 49 59. Vogel, J.M., Stearns, T., Rieder, C.L., and Palazzo, R.E. (1997). Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin. J Cell Biol 137 193 202. Wallin, K. L., Wiklu nd, F., Luostarinen, T., Angstrm, T., Anttila, T., Bergman, F., Hallmans, G., Ikheimo, I., Koskela, P., Lehtinen, M., et al. (2002). A populationbased prospective study of Chlamydia trachomatis infection and cervical carcinoma. Int J Cancer 101 371 374 Wandke, C., Barisic, M., Sigl, R., Rauch, V., Wolf, F., Amaro, A.C., Tan, C.H., Pereira, A.J., Kutay, U., Maiato, H., et al. (2012). Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis. J Cell Biol 198 847 863. Weihua, Z., Lin, Q., Ramoth, A.J., Fan, D., and Fidler, I.J. (2011). Formation of solid tumors by a single multinucleated cancer cell. Cancer 117 4092 4099. Wheelhouse, N., and Longbottom, D. (2012). Endemic and emerging chlamydial infections of ani mals and their zoonotic implications. Transbound Emerg Dis 59, 283 291. WHO (2001). Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. World Health Organizaion, Geneva (2001). 150 Wittmann, T., Hy man, A., and Desai, A. (2001). The spindle: a dynamic assembly of microtubules and motors. Nat Cell Biol. Zhong, G. (2011). Chlamydia trachomatis secretion of proteases for manipulating host signaling pathways. Front Microbiol 2 14. Zhong, G., Fan, P., Ji H., Dong, F., and Huang, Y. (2001). Identification of a chlamydial proteaselike activity factor responsible for the degradation of host transcription factors. J Exp Med 193 935 942. Zyss, D., and Gergely, F. (2009). Centrosome function in cancer: guilt y or innocent? Trends Cell Biol 19, 334 346.
92 BIOGRAPHICAL SKETCH Heather Brown was born in Athens, GA, but moved to Penn Yan, NY to complete primary and secondary school. Heather had the opportunity to spend time in her uncles cytomegalovirus lab, wher e her love of confocal microscopes started. She then attended University of Rochester where she explored all the different majors in biology, while working in a physiology lab, before settling on Microbiology and Immunology. She was accepted into a summer internship at Whitney Laboratory for Marine Bioscience the summer before senior year. This strengthened her resolve to get her Ph. D. After graduating college, she was admitted into the Interdisplinary Program in Biomedi cal Sciences at University of Flor ida. Heather Brown performed her graduate research studies under the supervision of Dr. Scott Grieshaber on the pathogenesis of Chlamydia After obtaining her Ph.D. Heather hopes to have a career in the basic biology of cancer.