1 PHOSPHOLIPID SIGNALING IN NEUTROPH IL CHEMOTAXIS: EVIDENCE OF A POSITIVE FEEDBACK LOOP By VED PRAKASH SHARMA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007
2 2007 Ved Prakash Sharma
3 Dedicated to my parents.
4 ACKNOWLEDGMENTS I wish to convey my sincere thanks and appr eciation to my mentor, Dr. Atul Narang, for his guidance, constant encouragement and cons tructive criticism. I would like to thank our collaborators, Dr. Gerry Shaw and Dr. Colin Sumners, for providing excellent laboratory facilities, and also for their guidance and discussion. I would also like to thank the members of my committee, Dr. Ranga Narayanan and Dr. Lewis Johns for their advice and availability. I take this opportunity to thank Dr. Orion Weiner, Ha rvard, for his support with the HL60 cell line. My group member, Dr. Karthik Subramanian, in itiated me into neutrophil chemotaxis experiments and taught me the in tricacies involved. This work would not have been possible without the constant guidance and help from him. I would like to thank my other group members, Dr. Shakti Gupta, Dr. Eric May, Jason, and other graduate students in the department for their support and friendship. I am very gratef ul to the members of the Shaw and Sumners lab, Cui, Silas, Rachael, Tom, Hong Wei, for their he lp and laboratory assistance. I owe many thanks to Saurabh, Lyle, Jan and Field for their help duri ng the course of the work. I am grateful to Tim Vaught, Microscopy Core Facilit y, for help with microscopy. Special thanks go to Priyank, Alok, Siva, V ijay, Kanchan, Satish, Chinki, Tushar, Murali, Ashish, Gunjan and all my other friends for thei r constant support and help. Finally, I want to express my deep gratitude and love for my pa rents, brother and sister for their unwavering support and encouragement.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........7 LIST OF ABBREVIATIONS.......................................................................................................... 8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 CHEMOTAXIS IN EUKARYOTIC CELLS.........................................................................11 Introduction................................................................................................................... ..........11 Identification of the First Polarized Component....................................................................13 Spatiotemporal Dynamics.......................................................................................................1 5 Unique Localization........................................................................................................15 Polarized Sensitivity........................................................................................................16 Adaptation and Spontaneous Polarization.......................................................................16 2 MECHANISTIC MODELS OF PHO SPHOINOSITIDE POLARIZATION........................23 Signaling Pathways in Chemotaxis........................................................................................23 The Positive Feedback Loop Signaling..................................................................................25 3 EXPERIMENTAL EVIDENCE FOR PI(4,5)P2 LOCALIZATION AND POSITIVE FEEDBACK SIGNALING IN NE UTROPHIL-LIKE HL-60 CELLS..................................30 The AM-212 Antibody Is a Speci fic Marker for PI(4,5)P2....................................................30 Optimization of the Immunostaining Protocols......................................................................31 The PI(3,4,5)P3 and PI(4,5)P2 Localize at the Leading Edge.................................................33 The PI5KI and PI5KI Colocalize with PI(4,5)P2 and PI(3,4,5)P3 at the Leading Edge.....34 The PI3K Inhibition Impairs the Localization of Both PI(3,4,5)P3 and PI(4,5)P2.................35 Evidence for PI4K-Dependent Positive Feedback Loop........................................................36 4 ACTIN-BINDING PROTEI N, CORONIN-1A IN NEUTROPHIL CHEMOTAXIS...........48 Generation of Coronin-1a Antibody.......................................................................................49 Characterization and Immunostain ing of Coronin-1a Antibody............................................50 5 CONCLUSIONS.................................................................................................................. ..54 Localization of PI(4,5)P2 at the Leading Edge.......................................................................54 Positive Feedback Loop Mechanism for PI(3,4,5)P3 Polarization.........................................55
6 APPENDIX MATERIALS AND METHODS............................................................................58 Materials...................................................................................................................... ...........58 Cell Culture and Differentiation.............................................................................................58 Dot Blot Assay................................................................................................................. .......58 Enzyme-Linked Immunosorbent Assay (ELISA)..................................................................59 Chemotaxis Assay............................................................................................................... ...59 Immunostaining and Microscopy...........................................................................................59 Plasma Membrane Labeling...................................................................................................60 Western Blotting............................................................................................................... ......60 LIST OF REFERENCES............................................................................................................. ..61 BIOGRAPHICAL SKETCH.........................................................................................................69
7 LIST OF FIGURES Figure page 1-1 Five phases of the chemotactic cycle................................................................................19 1-2 Signaling involved in gradient sensing.............................................................................19 1-3 Principle of the fluores cent imaging experiments............................................................20 1-4 Simple model for loca lization of phosphoinositides.........................................................20 1-5 Schematic representation of the spatiotemporal dynamics...............................................21 1-6 Dependence on pre-existing polarity................................................................................22 2-1 The phosphoinositide cycle...............................................................................................2 8 2-2 Kinetic scheme of the positive feedback loop..................................................................28 2-3 Components of the positive feedback loop in space and time..........................................29 2-4 Pathway for synthesis of PI(3,4,5)P3 and the various posit ive feedback loops...............29 3-1 The AM-212 antibody is a sp ecific marker for PI(4,5)P2................................................37 3-2 Immunostaining patterns of PI(4,5)P2 and PI5KI vary depending on the fixation and permeabilization protocols..........................................................................................39 3-3 Comparison of PI(3,4,5)P3 and PI(4,5)P2 immunostaining.............................................40 3-4 The localization of PI(4,5)P2 is not an artifact due to membrane folds...........................41 3-5 PI(3,4,5)P3 and PI5KI colocalize at the leading e dge of fMLP-stimulated HL-60 cells.......................................................................................................................... ..........43 3-6 PI(4,5)P2, PI5KI and PI5KI colocalize at the leading edge of fMLP-stimulated HL-60 cells.................................................................................................................... .....44 3-7 Effect of PI3K inhibition on PIP3 and PIP2 polarizations...............................................46 3-8 Effect of PI3K inhibition on PIP3 and PI4KII polarizations..........................................47 4-1 Nucleotide sequence of homo sapien s coronin 1A (CORO1A), (Genbank accession NM_007074)..................................................................................................................... .51 4-2 The pATH11 vector showing trpE gene...........................................................................52 4-3 Characterization and immunostain ing of rabbit coronin-1a antibody...............................53
8 LIST OF ABBREVIATIONS fMLP N-formyl-L-methionyl-L-leucyl-L-phenylalanine LPA lysophosphatidic acid LPC lysophosphatidylcholine PE phosphatidylethanolamine PC phosphatidylcholine PI3K phosphatidylinositol 3-kinase PI5K phosphatidylinositol 5-kinase PTEN phosphatase and tensin homolog S1P sphingosine-1-phosphate PA phosphatidic Acid PS phosphatidylserine PI phosphatidylinositol PI(4)P phosphatidylinositol 4-phosphate PI(3,4)P2 phosphatidylinositol 3,4-bisphosphate PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate PIP2 phosphatidylinositol 4,5-bisphosphate PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate PIP3 phosphatidylinositol 3,4,5-trisphosphate dHL-60 Differentiated HL-60 DMSO Dimethyl sulfoxide PDGF Platelet-derived growth factor
9 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHOSPHOLIPID SIGNALING IN NEUTROPH IL CHEMOTAXIS: EVIDENCE OF A POSITIVE FEEDBACK LOOP By Ved Prakash Sharma August 2007 Chair: Ranga Narayanan Major: Chemical Engineering The crawling movement of cells in response to a chemical gradient is a complex process involving the orchestration of various intracellular signaling mo lecules. Although a complete mechanism for this process remain s elusive, the very first step of gradient sensing, enabling the cell to perceive the direction of the imposed gradient, has become mo re transparent. The increased understanding of this st ep has been driven by the discove ry that application of a weak chemoattractant gradient results in the localiz ation of membrane phosphoinositides at the front end of the cell, which then act as a Â“compassÂ” for the forward motility of the cell. Cell migration plays a pivotal role in diverse biological phe nomena such as wound healing, cancer metastasis and inflammatory response. Therefore, an unde rstanding of the gradient sensing has vast applications in the treatmen t of numerous human diseases. Among the earliest events in the response of mo tile cells to a chemoattractant gradient is the localization of PtdIns(3,4,5)P3 at the leading edge. One m odel proposed to explain this localization is complementary regulation of PI3K and PTEN, which implies that in polarized cells, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 have reciprocal concentra tion profiles. To test the validity of the model, we studied the spatial distributions of PtdIns(4,5)P2, PtdIns(3,4,5)P3, PI5KI and PI5KI in fMLP-stimulated neutrophil-like HL-60 cells. We found that both
10 PtdIns(3,4,5)P3 and PtdIns(4,5)P2 localize sharply at the lead ing edge. Furthermore, PI5KI and PI5KI colocalize with PtdIns(4,5)P2 and PtdIns(3,4,5)P3. When PI3K was inhibited using a panPI3K inhibitor (LY294002) or PI3K inhibitor (IC87114), we found that not only PtdIns(3,4,5)P3 but also PtdIns(4,5)P2 polarization at th e leading edge is diminished. These results suggest that in neutrophil-like HL-60 cells, there is a PtdIns(3,4,5)P3-dependent positive feedback loop that stimulates the PI 5KI-mediated synthesis of PtdIns(4,5)P2. The data also suggests that complementary regulation of PI 3K and PTEN is not the sole or dominant mechanism of PtdIns(3,4,5)P3 polarization. Another kinase of the signaling pathway, PI4KII has been shown to translocate from the cytosol to the membrane ruffles in PDGF stimul ated HeLa cells. In neutrophil-like HL-60 cells, we found that PI4KII localizes at the leading edge and th e application of pan-PI3K inhibitor leads to diminished PI4KII polarization. This data suggest s the possibility of one more PtdIns(3,4,5)P3-dependent positive feedback loop in the PI3K signaling pathway. Coronin has been identified as a major me mbrane associated protein in migrating Dictyostelium discoideum. The protein is thought to function through its interaction with F-actin and Arp2/3 protein complex, which plays a role in generating bran ches in the actin filament network. One member of the family, Coronin-1a, is restricted in expression to cells of hematopoietic lineage. We generated a specific rabbit coronin-1a antibody and have shown that in neutrophil-like HL60 cells, coronin-1a immuno-re activity is concentrated at the leading edge, where it colocalizes with actin.
11 CHAPTER 1 CHEMOTAXIS IN EUKARYOTIC CELLS Introduction Directed cell migration accompanie s us from conception to death. At birth, controlled cell migr ation brings about shape and organization to the embryo (Bray, 1992). As the embryo develops, neurons na vigate to appropriate regions of the body and direct the formation of the nervous system. In an adult, cell migration is indispensable for the defe nse and maintenance of the body. Neutrophils rapidly detect in flammation sites and engulf i nvading pathogens (Jones, 2000). Fibroblasts under the skin move in larg e numbers towards wounded areas and induce healing (Martin, 1997). Uncontrolled cell migration can also acceler ate death. Cancer metastasis is caused by directed cell migration of tu mor cells from the primary site to preferential sites of metastasis (Moore, 2001). Thus, a good understanding of cell migration sh ould be able to shed light on all these biological phenomena. Most eukaryotic cells move by crawling on a surface. The crawling movement is initiated in response to an external stimulus, which is fr equently a chemical con centration gradient. The resultant motion propels the cell forward along the direction of highe st concentration. The chemical that induces the movement is called chemoattractant and the movement itself is called chemotaxis (Weiner, 2002c). Eukaryotic chemotaxis is cyclic and each cycle consists of five distin ct phases (Figure 1-1) (Lauffenburger and Horwitz, 1996). The cell first sens es the external chemoattractant gradient using specialized receptive proteins on its me mbrane. This results in the extension of a protrusion, called a pseudopod, by polymerizing actin in the direction of maximum chemoattractant. In order to c onvert this response to motion, th e pseudopod then adheres to the substratum. This is then followed by a contr action phase, where the cell body and nucleus are
12 pulled forward. The last step in locomotion consists of two distinct proce sses, deadhesion of the protrusion and retraction of the tail. Each phase of the chemotactic cycle is a co mplex process involving the coordinated action of a large constellation of signali ng molecules, many of which have been identified. Still lacking, however, is a synthetic theory explaining how these molecules are organized in space and time. This work is primarily concerned with understanding the first phase of the cycle gradient sensing the mechanism that enables the cell to read the external gradient and extend a pseudopod precisely at its leading edge the region exposed to the highest chemoattractant concentration. The chemoattractant gradients imposed in the extracellular space are often quite small (1Â– 2% concentration change over th e length of the cell) (Tranquil lo et al., 1988), but the actin polymers synthesized in response to the gradie nt are found exclusively at the leading edge (Coates et al., 1992; Hall et al., 1988). Thus, a key problem of gradient sens ing is the elucidation of the mechanism that mediates the formation of a highly polarized distribution of actin polymers in response to a relatively m ild chemoattractant gradient. Several cell types have been used as model systems for st udying gradient sensing. These include fast moving-cells such as neutrophils (Rickert et al., 2000) and the slime mould amoeba, Dictyostelium (Parent and Devreotes, 1999), and slow-m oving cells such as neurons (Song and Poo, 1999), budding yeast (Sohrmann and Peter, 2003; Wedlich-Soldner and Li, 2003) and fibroblasts (Haugh et al., 2000). Although several aspects of this work apply to slow moving cells, the primary focus will be on the fast-moving cell types. The chemoattractant gradient is transmitted to the actin polymerization machinery by a signal transduction pathway that starts with receptors on the cell surface and terminates in
13 proteins that catalyze actin polymerization. In Dictyostelium and neutrophils the receptors are generally, G-protein coupled receptors (GPCRs ). Upon ligand binding, th ese receptors activate heterotrimeric G-proteins and dissociate them into G and G subunits. Each of these subunits then activates a train of signaling events, which ev entually activates proteins that catalyze actin polymerization. It is, therefore, conceivable that actin polymers inherit thei r highly polarized distribution from some molecule that is ups tream of the polymers in the pathway. Hence, it becomes crucial to identify the first polarized component in the chemoattractant activat ed signal transduction pathway. Recent experiments (Haugh et al., 2000; Me ili et al., 1999; Parent et al., 1998; Servant et al., 2000) have Shown that membrane-resid ent phosphoinositides, phosphatidy linositol 3,4,5-trisphosphate (PI(3,4,5)P3 or simply PIP3) and phosphatidylinositol 3,4 -bisphosphate (PI(3,4)P2)are among the earliest polarized component s of the signal transduction pathway involved in gradient sensing.. Studied the spatiotemporal dynamics of th ese phospholipids in response to various chemoattractant profiles. Identification of the First Polarized Component Motile cells are transfected w ith chimeric proteins made by fusing a fluorescent protein either to the molecule of interest, or to a subs tance that binds specifica lly to the molecule of interest and thus Â“reportsÂ” on it. For example, the pleckstrin homology (PH) domain of Akt binds specifically to PI(3,4)P2 and PI(3,4,5)P3, so that the fusion protein GFP-PH-Akt reports on the distribution of these phosphoinositid es. A transfected cell is then exposed to a chemoattractant gradient by releasing chemoattractant from a micropipette and the resultant intracellular distribution of the fluorescent pr obes is visualized using fluorescence microscopy (Figure 1-3).
14 This approach allows the study of spatial polar ity at each stage of the signal transduction pathway. Specifically, the following has been found. Membrane receptors are not polarized. It was initially suggested that the localized actin polymerization could be explained by a no n-uniform distribution of receptors on the cell membrane (Cassimeris and Zigmond, 1990; Zigmond et al., 1981). However, recent studies have univocally demonstrated that wh en exposed to a chemoattractant gradient, receptors fail to redistribute and are in f act uniformly distributed (Servant et al., 1999; Xiao et al., 1997). Receptor occupancy and G-proteins are not significantly polarized Cells containing inactive G-proteins fail to chemotax(Jin et al ., 1998). Hence, receptors and G-proteins are essential for transmission of the chemoattractant signal. However, when cells are exposed to a chemoattractant gradient, the ligand bound receptors and active G-proteins show only a shallow anterior-posterior profile (Janetopoulos et al ., 2001; Ueda et al., 2001). It follows that receptors and G-proteins are requi red, but are not the source of the localized amplification observed at the leading edge of the cell. Phosphoinositides are significantly polarized Within 5 to 10 secs of applying a chemoattractant gradient, PH domains of si gnaling molecules such as Akt, CRAC, PhdA and Btk strongly polarize at th e edge of the cell membrane that received the strongest chemoattractant stimulus(Meili et al., 1999; Parent et al., 1998; Servant et al., 2000). These PH domains report specifically on the intracellular distri bution of membraneresident phosphoinositides, PI(3,4)P2 and PI(3,4,5)P3. In neutrophils, the gradient of these PH domains is nearly six times the chemoa ttractant gradient(Servant et al., 2000). It follows that these phosphoinositides are am ong the earliest polarized components of the signal transduction pathway. Hereafter, this phenomenon will be referred to as phosphoinositide localization Phosphoinositides polarize indepe ndent of the actin cytoskeleton. The phosphoinositide localization is tightly accomp anied with actin polymerization and pseudopod formation. This raised the possibility that actin polymerization is necessary for the translocation of phosphoinostides. In orde r to test this, a toxin called Latranculin A is added to depolymerize the actin and give the cell a rounded morphology. However, cells still recruited the phosphoinositides asymme trically to the face closest to the pipette (Parent et al., 1998; Parent and Devreotes, 1999; Servant et al., 2000) This showed that the localization of phosphoinositides takes pl ace independent of the actin cytoskeleton. Hence, the key problem in the study of grad ient sensing now becomes: What is the mechanism underlying the phosphoinositide localization? At first sight, the localization of phosphoinositid es seems explicable in terms of a simple amplification model. It suffices to postulate that the phosphoinositide synthesis responds to
15 receptor activation in a highly coope rative manner (Hill-type kinetics ) (Figure 1-4). In this case, the phosphoinositide distribution will be similar in shape, but steep er in slope, when compared to the chemoattractant concentration profile. In ot her words, the phosphoinositide distribution is an amplified version of the chemoa ttractant concentration profile. However, several experiments show that eukaryotic gradient sensing is not a matter of simple amplif ication. In each of the experiments below, the distri bution of the phosphoinositide lo calization differs from the chemoattractant profile imposed on the cell. Spatiotemporal Dynamics Motile cells are exposed to a variety of chemoattractant prof iles which include (1) steady or time varying gradients produced by rele asing chemoattractant from one or more chemoattractant sources (2) steady uniform profiles obtained by im mersing the cell in chemoattractant. These experiment s result in the manifestation of the following spatiotemporal dynamics (Figure 1-5): (1) unique localization (2) polar ized sensitivity (3) adaptation and (4) spontaneous polarization. In the following section, these dynamics will be defined precisely by describing the experiments and th e corresponding observations. Unique Localization In normal motile cells, only one leading edge ultimately develops, regardless of the external signal. Foxman et al., 1997, provided a convincing demonstration of this behavior by exposing a cluster of motile cells to two chemoa ttractant sources located at different distances from the cluster. All the cells migrated towards the closer source, showing that cells respond to the stronger stimulus and completely ignore the weaker stimulus. This phenomenon is referred to as unique localization.
16 Polarized Sensitivity If a cell that has localized phosphoinositides in a certain direction is exposed to a modest chemoattractant gradient along a different direct ion, a new localization does not develop at the point with the highest chemoattr actant concentration. Instead, the existing localization turns and reorients itself along the new grad ient. This phenomenon is called pol arized sensitivity, since the turning response suggests that pr e-existing edge or Â“poleÂ” is more sensitive to chemotactic signals than all other regions of the cell. Interestingly, if the new chemoattractant gradient is relatively large and localized, the existing pseudopod retracts and a new one grows along the direction of the new gradient. The induction of a turning response depends not only on the external concentration, but also on the preexisting polarity of the cell. This is dramatically illustrated by an experiment shown in Figure 1-6 (Devreotes and Janetopoulos, 2003). The upper panel shows three cells labeled a,b and c moving toward the chemoattractant so urce shown as pipette 2. Interestingly, even though cell b is much closer to pipette 2 than cell a it does not respond at all, whereas the more distant cell a turns toward pipette 2. This shows that the cells b and c that are closer to pipette 1, and hence, likely to be more polarize d, are less responsive to th e subsequent influence of pipette 2. Adaptation and Spontaneous Polarization When resting cells are stimulated with a uniform chemoattractant concentration, the phosphoinositides in the membrane increase uniform ly within 5Â–10 seconds. However, if this uniform concentration is maintain ed at the same level, the phos phoinositides decrease to their prestimulus steady state (Parent et al., 1998; Parent and Devreotes, 1999). This phenomenon is called adaptation (Othmer and Schaap, 1998). The mechanism of adaptation is only partially understood.
17 In Dictyostelium earlier work showed that adaptati on was perfect, i.e. the membrane phosphoinositides returned to their basal steady stat e (Parent et al., 1998). However, recent work shows that cells frequently form multiple pa tches of phosphoinositide lo calization instead of completely adapting to their prestimulus state, suggesting that adaptation is in fact, imperfect (Postma et al., 2003). Adapatation is manifested in a slightly different manner in ne utrophils (Zigmond and Sullivan, 1979). After exhibiting a uniform initia l increase, the phosphoinositides spontaneously localize at a random location on the membrane a nd decrease at all othe r regions of the cell (Servant et al., 2000; Wang et al., 2002). The region of phosphoinositide localization subsequently becomes the leading edge of the cell. This phenomenon is called spontaneous polarization to emphasize the fact that the phosphoinositides and the cell morphology polarize even though the chemoattractant environment is macroscopically uniform (Wedlich-Soldner and Li, 2003). The time taken for the uniform initial in crease is independent of the chemoattractant concentration (Zigmond and Sullivan, 1979). However, the amplitude of the initial response and the time taken for spontaneous polarization is directly proportional to the chemoattractant concentration. This dissertation has been organized in th e following manner. Chapter 2 summarizes the signaling pathways involved in th e gradient sensing mechanism and in particular, the role of the positive feedback signaling in phosphoinositide polarization. The comparison of positive feedback model with other pr evalent models has also been described. Chapter 3 provides experimental evidence in support of positive feed back loop mechanism in neutrophil-like HL60 cells. Of particular interest is the finding that phosphatidy linositol 4,5-bisphosphate (PI(4,5)P2 or simply PIP2) localizes at the leading e dge of the chemoattractant stim ulated neutrophil-like HL60
18 cells. Chapter 4 describes the development and ch aracterization of an an tibody to coronin-1a, an actin binding protein, which has been implicated in Dictyostelium chemotaxis (de Hostos et al., 1991). We show that in neutroph il-like HL60 cells, coronin-1a localizes at the leading edge.
19 Figure 1-1. Five phases of the chemotactic cycl e (adapted from (Lauffenburger and Horwitz, 1996) ) (1) Gradient sensing (2) Protrusi on of pseudopod (3) Adhesion to substratum (4) Traction of the cell body and (5 ) Retraction of the cell tail. Figure 1-2. Signaling involved in gradient sensing. The chemoattr actant binds to G-protein coupled receptors on the cell surface, which in turn induces the dissociation of Gproteins. Active G-proteins then propagate the signal downstream, eventually resulting in localized actin polymerization at the leading edge of th e cell. In spite of a shallow external gradient, the response obs erved is highly localized at the leading edge of the cell.
20 Figure 1-3. Principle of the fluor escent imaging experiments. Figure 1-4. Simple model for loca lization of phosphoinosi tides. The activity of the enzyme that synthesizes phosphoinositides is a sharp non-li near function of the active receptors. Any shallow chemoattractant gradient cen tered around the threshold would yield a large phosphoinositide gradient. This model shows that the phos phoinositide response is simply an amplified version of the chemoattractant gradient.
21 Figure 1-5. Schematic representation of the spat iotemporal dynamics. (A) When a resting cell (shown in 1) is exposed to a uniform in crease in chemoattractant concentration, it immediately responds by increasing phos phoinositides all over the membrane (shown in 2). This is followed by the spontan eous polarization (shown in 3) of phosphoinositides along a random direction. (B) Wh en a resting cell (shown in 4) is exposed to a gradient of chemoattractant it accumulates phos phoinositides at the point of highest chemoattractant concentra tion (shown in 5). When the direction of the gradient is switched the existing locali zation turns in the direction of the new source of chemoattractant (shown in 6) This phenomenon is called polarized sensitivity. (C) When a resting cell (s hown in 7) is exposed to two unequal chemoattractant sources, it responds to bot h sources initially. However, as time progresses the response to the stronger sour ce grows and the response to the weaker source is completely abolished. We re fer to this as unique localization.
22 Figure 1-6. Dependence on preexisting polarity. Response of three cells labeled a b and c to sequential stimulation by two chemoattr actant sources (from (Devreotes and Janetopoulos, 2003)). The top panel shows all three cells moving towards the sole chemoattractant source (pipette 1). The bottom panel shows the response when an additional source (pipette 2) is turned on some time later. Cell Â‘aÂ’ immediately turns toward pipette 2, whereas cells Â‘bÂ’ and Â‘cÂ’ show no response at all.
23 CHAPTER 2 MECHANISTIC MODELS OF PHO SPHOINOSITIDE POLARIZATION The desire to capture the spatiotemporal dyna mics of gradient sensing has spurred the development of several mathematical m odels. Three of these models contain a short-range activator that is synthesized autocatalytically, and a long-range inhibitor that inhibits the synthesis of the activator (Meinha rdt, 1999; Narang et al., 2001; Po stma et al., 2001). They differ only with respect to the postu lated mechanisms of the activ ation and inhibition. Two other models contain a long-range inhib itor but no activator, i.e., there is no autocatalytic synthesis (Krishnan and Iglesias, 2003; Levchenko and Ig lesias, 2002; Rappel et al., 2002). These two models differ with respect to the reaction kinetics Â— the synthesis rate of the inhibitor is rapid in the first case, and slow in the second case. R ecently, (Haugh et al., 2004; Schneider et al., 2004) have proposed a model based on the turnover an d diffusion of a single intracellular component. Signaling Pathways in Chemotaxis The signaling pathway that follows receptor activation is the subject of ongoing research. In Dictyostelium discoideum and neutrophils, receptor-ligand binding activates heterotrimeric Gproteins. Activated G-proteins can activate PI3K by direct binding (Stephens et al., 1997), resulting in the synthesis of PIP3 from PIP2 (see Figure 2-1). The PIP3 thus produced and the G subunit then synergisti cally activates the membrane-reside nt protein, P-Rex1 (Weiner, 2002a; Welch et al., 2002; Welch et al., 2003), which be longs to the Dbl family Rac-GEFs (guaninenucleotide exchange factors for Rac) that ac tivate Rac. Activated P-Rex1 then converts the inactive Rac-GDP to active Rac-GTP. There is growing evidence that Rac mediates both PI3K and PI5K (Bokoch et al., 1996; Tolias et al., 2000). This has le d to the suggestion that PIP3 and Rac function in a positive feedback loop for more synthesis of PIP2 and PIP3 (Narang et al., 2001; Srinivasan et al., 2003;
24 Weiner et al., 2002). Activation of PI5K creates yet another positive feedback loop because this increases the synthesis rate of PIP2 and its downstream product, phos phatidic acid (PA), a potent activator of PI5K (Ishihara et al., 1998). Becau se of these two positive feedback loops, the synthesis rate of PIP2 and PIP3 can rapidly accelerate to high levels. Such high synthesis rates of PIP2 and PIP3 can be sustained for no more than a second because the concentration of phos phatidylinositol (PI) in the plas ma membrane is quite small (Willars et al., 1998). Depletion of PI in the plas ma membrane is prevented by the cytosolic PI transport protein (PITP), which tr ansfers readily available PI fr om the endoplasmic reticulum to the plasma membrane (Cockcroft, 1999).The PIP2 formed by successive phosphorylation of PI is hydrolyzed by PLC to diacylglycerol (DG) and cytosolic inositol 1,4,5-triphosphate (IP3). Diacylglycerol is converted to PA and transferred to the endopl asmic reticulum for regeneration of PI. Inositol produced by rapid dephosphorylation of IP3 via multiple pathways (Berridge and Irvine, 1989), also particip ates in PI regeneration. Several observations suggest a causal link between PIP2 (Honda et al., 1999; Tall et al., 2000; Watt et al., 2002)/ PIP3 (Meili et al., 1999; Parent et al ., 1998; Servant et al., 2000) synthesis and lamellipod extension. Lamellipods ar e formed precisely at the same location as PIP2 (Tall et al., 2000)/PIP3(Parent et al., 1998; Servant et al., 2000) production. The enzymes that catalyze the synthesis of these phosphoinos tides, PI3K (Funamoto et al., 2002) and PI5K (Doughman et al., 2003b), are also recruited to the leading edge of the cell. In addition, it has been shown that PIP2, in conjunction with GTP-bound Cdc42, is a strong activator of N-WASP, which in turn activates Arp2/3 (Zigm ond, 2000). Similarly, Rac activation and PIP3 production activate WAVE proteins, which can also ac tivate Arp2/3 (Zigmond, 2000). Activated Arp2/3 mediates actin polymerization by nuc leating the sides of pre-existi ng actin filament s (Pollard et
25 al., 2000). Actin polymerization by Arp2/3 is believed to drive lamellipod protrusion (Borisy and Svitkina, 2000). Taken togeth er, these facts suggest that the localization of PIP2 and PIP3 resulting from the gradient sensing mechanism play s a crucial role in the subsequent extension of the lamellipod. The Positive Feedback Loop Signaling Although there is evidence in support of pos itive feedback signaling in neutrophil chemotaxis (Weiner et al., 2002), the components of this signaling pa thway are yet to be identified. Based on the current literature, on e can construct the model shown in Figure 2-2. In neutrophils, the receptors for the chem oattractant fMLP are coupled to membraneresident G-proteins (Figure 2-2). When a chemoa ttractant molecule binds to a receptor, the Gprotein coupled to the receptor dissociates into its G i and G subunits. The G via its p101 subunit activates PI3K subunit, resulting in the synthesis of PIP3 (Brock et al., 2003; Stephens et al., 1997). The PIP3 subunit then synergistically activat es the membrane-resident protein, PRex1, which belongs to the Dbl family of Rac-GE Fs (guanine-nucleotide exchange factors for Rac) that activate Rac (Weiner, 2002b; Welch et al., 2003). In resti ng cells, Rac is cytosolic and GDP-bound. This state is maintain ed through its interaction with a GDP dissociation inhibitor (GDI) (Figure 2-3). Upon receptor activa tion, GDI dissociates from Rac by an unknown mechanism, and Rac-GDP targets to membrane vi a its C-terminal prenyl ation, where Rac-GEFs, such as P-Rex1, activate Rac by dissociating GDP and binding GTP (Welch et al., 2003). To be sure, other Rac-GEFs of the Dbl family could al so activate Rac, but P-Rex1 is most important insofar as its activity represents 65% of the total Rac-GEF activity in neutrophils. In vitro experiments have shown that R ac interacts with the p85 subunit of PI3K (Bokoch et al., 1996). This has led to the suggestion that PIP3 and Rac function in a positive feedback
26 loop leading to rapid accumulation of PIP3 (Rickert et al., 2000; Sadhu et al., 2003; Weiner et al., 2002). However, in vivo data show that less than 1% of the total PI3K activity can be attributed to Rac-PI3K (Tolias et al., 1995). A more plausible model for the closure of th e positive feedback loop is the Rac-mediated activation of the enzyme PI5K, wh ich catalyzes synthesis of PIP2 from PI4P. Both GDP-bound and GTP-bound Rac interact with PI5K via thei r C-terminus (Doughman et al., 2003b; Tolias et al., 2000; van Hennik et al., 2003; Weernink et al., 2004). Ho wever, only GTP-bound Rac can activate PI5K(Tolias et al., 2000; Weernink et al., 2004). Based on these facts, the following model is proposed (see Figure 2-2, Figure 23). Under resting conditions, a Rac-GDP/PI5K complex exists in the cytosol. Upon receptor ac tivation, this complex gets recruited to the leading edge where Rac-GEFs convert Rac-GDP /PI5K to Rac-GTP/PI5K. Rac-GTP may either activate PI5K or provide PI5K access to its plasma membrane substrate, PI4P. This results in localized synthesis of PIP2 at the plasma membrane. This increase in PIP2 drives the formation of more PIP3 and closes the feedback loop. The forgoing model for the positive feedback loop is supported by several observations. Introduction of membrane permeant PIP3 into resting neutrophils triggers the spontaneous polarization of endogenous PIP3 (Weiner et al., 2002). Howeve r, if PI3K is inhibited, membrane-permeant PIP3 polarization is inhibited. This implies that a functional positive loop is required for polarization, and PI3K is an essential component of this loop. Inhibition of PI3K results in lo ss of directionality, loss of PIP3 localization and improper chemotaxis of neutrophils (Hannigan et al ., 2002; Sadhu et al., 2003; Wang et al., 2002). Active Rac-GTP localizes at the leading edge of polarized neutroph ils (Gardiner et al., 2002). Overexpression of Rac-GTP results in PIP3 accumulation all over the plasma membrane (Srinivasan et al., 2003). In contrast, Rac-GDP inhibits PIP3 accumulation all over the plasma membrane. (Srinivasan et al., 2003).Hence, Rac activa tion is necessary and sufficient for PIP3 synthesis.
27 In human fibroblasts, PI5KI is cytosolic in serum-starved cells, but localizes to membrane ruffles upon growth factor stimulation (Dough man et al., 2003b). Inhibition of either PI5KI or PI3K completely abolishes ruffle formation. Overexpression of Rac-GTP and PI5KIinteraction inhibits the extensive ruffling phenotype. Sim ilar effects of PI5K on actin polymerization have been observed in bl ood platelets (Tolias et al., 2000). These observations are consistent w ith our model where PI3K, PI5K and Rac are essential for positive feedback. In vitro experiments show that coexpression of PI5K and Rac-GTP markedly stimulates PI5K activity, whereas coexpression of PI5K and Rac-GDP inhibits PI5Kactivity (Chatah and Abrams, 2001; Weernink et al., 2004). In vi vo studies show that PI5K and Rac-GTP colocalize at the plasma membrane and induce extensive actin polymer ization. In contrast, PI5K and GDP-bound Rac fail to localize at th e membrane and completely inhibit actin polymerization (Chatah and Abrams, 2001). Thus Rac regulates both the activity and the intracellular distri bution of PI5K. Recent experiments have isolated a novel PI5K homolog called PIPKH (Chatah and Abrams, 2001). This homolog does not possess PI5K activity, but acts as a scaffolding protein that binds to PI5Ks. Upon overexpression, PIPKH results in small increases in total PIP2 levels and dramatic increases in PIP3 levels. Consistent with the idea proposed in our model, the authors suggest that PIPKH recruits PI5K enzymes to specif ic intracellular sites where localized PIPsynthesis induces massive accumulation of PIP3. In HL60 cells, the C-terminus of Rac1 inte racts with PI5K (van Hennik et al., 2003). Introduction of the purified, membrane-permean t Rac1 C-terminus (containing no effector domain) inhibits the migration of HL60 cells. Consistent with our model, this inhibition occurs because the endogenous PI5K is satu rated with inactive Rac1 C-terminus, and therefore cannot synthesize PIP. In contrast, when a C-terminal Rac1 mutant that does not bind to PI5K is introduced into HL60 cells, mo tility is normal. This observation highlights the importance of the Rac1-PI5K interaction in the positive feedback loop. Recently Wei et al., 2002, have shown that in HeLa cells, upon PDGF stimulation, PI4KII but not PI4KII translocates from the cytosol to the membrane ruffles in the plasma membrane. They also found that overexpressi on of constitutively active RacV12 induces membrane ruffling and PI4KII translocation, whereas domina nt negative RacN17 blocks PI4KII translocation. This data sugge sts the possibility of anothe r positive feedback loop from PIP3 to PI4KII to further enhance PIP2 synthesis. Figure 2-4 is the simplified scheme of proposed positive feedback loops. The experiment s described in chapter 3 investigate these PI5Kand PI4Kmediated positive feedback loops.
28 Figure 2-1. The phosphoinositide cycle. The positiv e feedback loop has been shown in red. The inositol-mediated inhibition of phosphoi nositides has been shown in blue. Figure 2-2. Kinetic scheme of the positiv e feedback loop (shown in red).
29 Figure 2-3. Components of the positive feedback loop in space and time. Figure 2-4. Pathway for synthesis of PI(3,4,5)P3 and the various positiv e feedback loops. The figure shows only those isoforms of PIP kinases which appear to catalyze the appropriate phosphorylation reaction at th e leading edge. These include PI4KII (Wei et al., 2002), PI5KI (Doughman et al., 2003), PI5KI (Wang et al., 2004), PI3K (Li et al., 2000), and PI3K (Sadhu et al., 2003). Also shown in the figure is the reaction catalyzed by th e 3Â’-phosphatase, PTEN, which plays a central role in models of PI(3,4,5)P3 polarization based on the complementary action of PI3K and PTEN. The dashed lines (pathways 1, 2, 3) show the possible pathways of PI(3,4,5)P3-dependent positive feedback loops. PI4PPI4,5P2PIPI3,4,5P3PI5KI PI3K PI4KII Rac 1 2 3 PTEN PI4PPI4,5P2PIPI3,4,5P3PI5KI PI3K PI4KII Rac 1 2 3 PTEN
30 CHAPTER 3 EXPERIMENTAL EVIDENCE FOR PI( 4,5)P2 LOCALIZATI ON AND POSITIVE FEEDBACK SIGNALING IN NEUTROPHIL-LIKE HL-60 CELLS In this chapter we investigate the role of PI5KI -mediated positive feedback loop in the gradient sensing mechanism. As a model system we chose HL-60 cells, a human promyelocytic leukemia cell line, to perform the experiments. When treated with dime thyl sulfoxide (DMSO) for 5Â–6 days, HL-60 cells differentiate into ne utrophil-like cells. They look and behave like neutrophils, orient their polarity in response to the chemoattractant, fMLP, and migrate toward the highest chemoattractant concen tration (Hauert, 2002; Servant et al., 2000). The differentiated HL-60 cells, referred to hereafte r as dHL-60 cells, are easier to culture than the terminally differentiated neutrophils. Moreove r, dHL-60 cells have been the subject of numerous gradient sensing studies, providin g established benchmarks to test the validity of protocols used in this work. Details of all the experimental pr otocols are mentioned in the appendix A. The AM-212 Antibody Is a Sp ecific Marker for PI(4,5)P2 The monoclonal antibodies, KT-10 (Fukami et al., 1988) and AM-212 (Miyazawa et al., 1988), are the two most commonly used anti-PI(4,5)P2 antibodies. To check the specificity of these antibodies against various phospholipids, we performed dot blot assay with PIP strip (Echelon Biosciences, Salt Lake City, UT), wh ich has 15 different phospholipids spotted on a nitrocellulose membrane (Figure 3-1 A). The dot blot assay shows that the KT-10 antibody has a surprisingly high binding for PI(3,4)P2, and relatively low binding for PI(4,5)P2 and PI(3,4,5)P3 (Figure 3-1 B). In contrast, the AM-212 an tibody binds almost entirely to PI(4,5)P2, with only a marginal reactivity with PI(3,4,5)P3 (Figure 3-1 C). The cros s-reactivity with various phospholipids, including all other p hosphoinositides, is negligibly sma ll. Intensity an alysis of the blots showed that ~70% of th e KT-10 binding is for PI(3,4)P2, whereas ~85% of the AM-212 binding is for PI(4,5)P2. Since PI(4,5)P2 levels are 10-1000 times PI(3,4,5)P3 levels under all
31 conditions (stimulated or unstimulated), the antibody AM-212 can be considered a specific marker for PI(4,5)P2. In addition to using PI P strips from Echelon, we also spotted various phospholipids on nitrocellulose membrane and did th e dot blot assay. We obt ained results similar to those obtained with the commercial PIP stips. As a further test of AM-212 specificity, we also used enzyme-linked immunosorbent assay (ELISA) to check the binding of AM-212 anti body with the following four phospholipids: PI(4,5)P2, PI(3,4,5)P3, PI(3,4)P2 and PI(4)P. Again, AM-212 showed a strong affinity for PI(4,5)P2, slight cross-reac tivity with PI(3,4,5)P3, and marginal cross-reactivity with PI(3,4)P2 and PI(4)P (Figure 3-1 D). All th e immunostaining data for PI(4,5)P2 presented in this manuscript were generated with the AM-212 antibody. Optimization of the Immunostaining Protocols In the literature, many different fixing and permeabilization protocols have been used, resulting in diverse immunos taining patterns for PI(4,5)P2 and PI5KI Several studies have reported that PI(4,5)P2 is detected on the plasma and sub cellular membranes (Chen et al., 2002; Doughman et al., 2003b; Laux et al., 2000; Wang et al., 2004). Yet others have observed PI(4,5)P2 and PI5KI primarily in the nucleus (Boronenkov et al., 1998). In order to identify the effect of the fixing and permeabilization prot ocol on the staining pattern, we performed systematic studies with two fixatives (4% pa raformaldehyde, 4% paraformaldehyde + varying amounts of glutaraldehyde) and two de tergents (Triton X-100, digitonin). When we used 4% paraformaldehyde (PFA ) as the fixative and Triton X-100 as the detergent, there was no signal at the plas ma membrane. Instead, we observed punctate distributions of PI(4,5)P2 and PI5KI in the nucleus (Figure 3-2, upper panel) similar to those observed by Boronenkov et al. The lite rature suggests that this is due to the harshness of Triton
32 X-100. Hannah et al compared the st aining patterns of several proteins with PFA as the fixative, and Triton X-100 or digitonin as the detergents (Hannah et al ., 1998). In many cases, only the membrane was stained when digitonin was use d, whereas Triton X-100 resulted in markedly reduced membrane staining and pronounced in tracellular staining. It was concluded that digitonin permeabilizes only the plasma memb rane, whereas Triton X-100 solubilizes a significant part of the plasma membrane and permeabilizes the subcellular membranes. Our experiments support these observations. Indeed, when we replaced Triton X-100 with digitonin, anti-PI(4,5)P2 and anti-PI5KI antibodies stained only the plasma membrane (Figure 3-2, middle panel). Although the use of digitonin preserve d the membrane signal, the PI(4,5)P2 and PI5KI immunostaining consisted of patchy distributions at the plasma me mbrane. We reasoned that this was because PFA fixation failed to produce comple te cross-linking of th e proteins. Nakamura has shown that significant amounts of proteins ar e extracted when the cells are fixed with 4% PFA for as long as 30 mins (Nakamura, 2001). In c ontrast, almost no protei ns are extracted when 4% PFA is supplemented with 0.05% or 1% gl utaraldehyde (GTA). Th e addition of GTA is thought to accelerate protein cr oss-linking due to the presence of additional aldehyde groups (Kiernan, 2000). When we used 4% PFA + 0.05% GTA as the fixative, the PI(4,5)P2 and PI5KI staining became contiguous, and gr adients could be discerned even if Triton X-100 was used as the detergent (Figure 3-2, lower panel). The addition of GTA to PFA unmasked the PI(4,5)P2 and PI5KI gradients, but there was significant intracellular staining. We inferred that this is par tly due to the autofluorescence caused by unreacted aldehyde groups of GTA, and partly due to the permeabilization of intracellular membranes by Triton X-100 (result ing in especially pronounced intracellular
33 staining of PI5KI Figure 3-2 J). We mitigated the first problem by using the lowest concentration of GTA (0.05%) that yielded contiguous, rather th an patchy, distributions, and eliminated the latter problem by reverting to the use of digitonin as the permeabilizing agent. Figures 3-6A and 3-6B show that the optimi zed combination consisting of 4% PFA + 0.05% GTA as fixative and 10 g/mL digitonin as permeabilizing agen t yields sharp gradients of both PI(4,5)P2 and PI5KI All immunostaining data describe d below was generated using this optimized combination. PI(3,4,5)P3 and PI(4,5)P2 Localize at the Leading Edge The spontaneous polar ization of PI(3,4,5)P3 in HL-60 cells subjected to a uniform fMLP stimulus is well established (Weiner et al., 2002). We confirmed the existence of such PI(3,4,5)P3 localization in our experime nts (Figure 3-3 A). We were particularly interested in studying the distribut ion of PI(4,5)P2. We found that in contrast to the prediction of the CR model, PI(4,5)P2 also localizes at the leading edge (F igure 3-3 B). Importantly, the intensity gradient of PI(4,5)P2 is comparable to that for PI(3,4,5)P3. Hence, a significant gradient of PI(4,5)P2 develops despite its high basal levels. It has been previously obse rved that phosphoinositide loca lization can be an artifact resulting from membrane folding (van Rheenen a nd Jalink, 2002). To check if this is the case, we looked at the distribution of the membrane ma rker, DiO, in polarized HL-60 cells (Figures 34 A-D) and compared it with anti-PI(4,5)P2 immunostaining of HL-60 cells (Figures 3-4 F-I). The intensity profiles along the periphery of the DiO-stained cells were more or less uniform (Figure 3-4 E), whereas t hose of the anti-PI(4,5)P2-stained cells had a marked maximum at the leading edge (Figure 3-4 J). We also looked at the distributi on of an alternative lipophilic
34 membrane marker, DiI. We found a similar near -uniform distribution along the cell periphery. Thus, the localization of PI(4,5)P2 is not an artifact due to membrane folding. We also tried membrane dye and anti-PI(4,5)P2 costaining but these experiments were not successful. We found that the trea tment of cells with membrane dye interferes with PI(4,5)P2 as well as PI(3,4,5)P3 immunostaining. When exposed to uniform fMLP stimulus, cells treated with membrane dye, polarize and look morphologically si milar to non-treated cells, but they fail to stain for PI(4,5)P2 or PI(3,4,5)P3 antibodies. PI5KI and PI5KI Colocalize with PI(4,5)P2 and PI(3,4,5)P3 at the Leading Edge We also studied the spatial distribution of type I PI5-kinases. Although there are three isoforms of PI5KI (designated I I and I ), the evidence sugge sts that only the 1 and 1 play a role in membrane ruffling and motility. Doughma n et al studied the distribution of PI5KI and PI5KI in resting and PDGF-stimulated MG-63 fibr oblasts (Doughman et al ., 2003b). In resting cells, endogenous PI5KI and PI5KI were detected in the cyto sol and perinuclear vesicular structures, respectively. Upon PDGF stimulation, PI5KI translocated to the ruffles, whereas the distribution of PI5KI remained unchanged. Thus, the 1 but not the I isoform plays a role in membrane ruffling. Insofar as I isoform is concerned, there are two splice variants, namely, PI5KI 87 and PI5KI 90 (Doughman et al., 2003a) Recent experiments show that the 87 splice variant is enriched at the plasma membrane, and is a major contributor to the synthesis of the PI(4,5)P2 pool involved in GP CR-mediated Ins(1,4,5)P3 production (Wang et al., 2004). Thus, we were led to consider the spatial distributions of the I and I isoforms. We found that when HL-60 cells are stimulat ed with a uniform fMLP stimulus, PI5KI and PI5KI colocalize with PI(3,4,5)P3 (Figure 3-5) and PI(4,5)P2 (Figure 3-6) at the leading edge of the cells. The overlap was essentially perfect. These re sults corroborate the PI(4,5)P2
35 localization observed in the previous experiment, since the recruitment of PI5KI and PI5K to the leading edge is likely to cau se enhanced synt hesis of PI(4,5)P2 at the leading edge. The colocalization of PI(4,5)P2 and PI5Ks with PI(3,4,5)P3 at the leading edge, implicate the possible role of positive feedback l oop 2 (Figure 2-4) in PI(3,4,5)P3 polarization. PI3K Inhibition Impairs the Localization of Both PI(3,4,5)P3 and PI(4,5)P2 It has been previously shown that when HL-60 cells are pretreated with 100 M LY294002 (a pan-PI3K inhibitor, Vla hos et al., 1994), fMLP-induced PIP3 polarization is markedly reduced, and pretreatment with 300 M LY294002 abolishes the PIP3 polarization (Wang et al., 2002). We studied the effect of LY294002 pretreatment on the polarization of PI(4,5)P2. In fMLP stimulated human neutrophils, 50 M LY294002 completely inhibits the PI3K activity but doesnÂ’t significantly inhibit serine/thr eonine kinases, tyrosine kinases, lipid kinases and ATPase (Vlahos et al., 1994). So we chos e this value of LY294002 concentration to study the effect of PI3K inhibition on PIP3 and PIP2 polarizations in dHL-60 cells. To this end, dHL-60 cells, pretreated with 50 M LY294002, were exposed to a uniform fMLP (1 M) concentration. We found that consistent w ith earlier results, the fMLP -induced polarization of PIP3 at the leading edge is diminished (Figure 3-7B ). Interestingly, pretreatment with 50 M LY294002 also abrogated the polarization of PIP2 at the leading edge (Figure 3-7E). Neutrophils contain members of class I PI3K s, which are subdivided into class IA ( and ) and class IB ( ). Both classes have same catalytic subunit p110 but the mode of regulation is different. The class IB enzyme (PI3K ) has a p101 regulatory subunit and is activated by Gprotein-coupled receptors. Th e class IA enzymes (PI3K ) have p55-85 regulatory subunits and are classically activated by tyrosine ki nase-coupled receptors. In neutrophils, PI3K and
36 PI3K have been implicated in the fMLP-stimulated synthesis of PIP3 (Condliffe et al., 2005; Li et al., 2000; Sadhu et al., 2003). To test the role of PI3K inhibition on PIP3 and PIP2 polarizations, we performed experiments with PI3K inhibitor (IC87114), which was a kind gift from ICOS corporation. At 25 M, IC87114 inhibits PI3K but does not significantly affect other PI3K isofoms or other kinases (Sadhu et al., 2003). We found that, similar to LY294002 experiment results, 25 M IC87114 diminishes not only PIP3 but also PIP2 polarizations at the leading edge in fMLP-stimulated dHL-60 cells. The abrogation of PIP2 polarization, by PI3K inhibitors (LY294002 and IC87114), suggest that loop 2 in Figure 2-4 is a PIP3-dependent positive feedback loop. Evidence for PI4K-Dependent Positive Feedback Loop Recently Wei et al., 2002, have shown that in HeLa cells, upon PDGF stimulation, PI4KII but not PI4KII translocates from the cytosol to the membrane ruffles in the plasma membrane. They also found that overexpressi on of constitutively active RacV12 induces membrane ruffling and PI4KII translocation, whereas domina nt negative RacN17 blocks PI4KII translocation. This data sugge sts the possibility of anothe r positive feedback loop from PIP3 to PI4KII to further enhance PIP2 synthesis. We investigated this positive feedback loop in neutrophil-like HL-60 cells. Immunosta ining experiments s howed that PI4KII localizes at the leading edge of these cells, where it colocalizes with PIP3 (Figure 3-8, upper panel). When we blocked PI3K using 50 M LY294002, we found that not only PIP3, but also PI4KII polarization is diminished (Figure 3-8, lower panel). This preliminary data suggests the possibility of one more PIP3-dependent positive feedback lo op 3 (Figure 2-4) to activate PI4KII leading to enhanced production of PIP2 and ultimately PIP3 localization at the leading edge of neutrophillike HL-60 cells.
37 Figure 3-1. The AM-212 antibody is a specific marker for PI(4,5)P2. Dot blot assay was done on PIP strips (Echelon Biosciences, Salt Lake city, UT) to check the specificity of two commonly used PI(4,5)P2 antibodies, KT-10 (21) and AM-212 (22), against 15 phospholipids. (A) Schematic diagram of a PIP strip showing all the phospholipid spots: 1. LPA; 2. LPC; 3. PI; 4. PI(3)P; 5. PI(4)P; 6. PI(5)P; 7. PE; 8. PC; 9. S1P; 10. PI(3,4)P2; 11. PI(3,5)P2; 12. PI(4,5)P2; 13. PI(3,4,5)P3; 14. PA; 15. PS; 16. Blank. (B) KT-10 binds primarily to PI(3,4)P2, and exhibits slight cros s-reactivity with PI(4,5)P2 and PI(3,4,5)P3 (C) AM-212 binds speci fically to PI(4,5)P2, and very slightly with PI(3,4,5)P3. The data is representative of 5 i ndependent dot blot experiments for each of the antibodies. (D) A 96-well polystyren e microtiter plate wa s coated with the following four phospholipids: PI(4,5)P2, PI(3,4,5)P3, PI(3,4)P2 and PI(4)P. The detection of antibody-antigen interaction using ELISA wa s done as described. Again, AM-212 shows strong reac tivity with PI(4,5)P2 with slight cross-reactivity with PI(3,4,5)P3. ELISA data is representative of 3 independent experiments.
38 Figure 3-1. Continued PI(3,4)P2 PI(4,5)P2 PI(3,4,5)P3 PI(4,5)P2 PI(3,4,5)P3 A Schematic B KT-10 C AM-212 PtdIns(4,5)P2 PtdIns(3,4,5)P3 PtdIns(4)P PtdIns(3,4)P20.0 0.4 0.8 1.2 1.6 100.0 50.0 25.0 12.5 6.3 3.1 PI(4,5)P2 PI(3,4,5)P3 PI(3,4)P2 PI ( 4 ) P Serial AM-212 dilution ( l ) A405 D
39 Figure 3-2. Immunostaining patterns of PI(4,5)P2 and PI5KI vary depending on the fixation and permeabilization protocols.Differentiated HL-60 cells were plated on glass coverslips and then stimulated with 1 M fMLP for 2 mins. A variety of fixative and permeablizing agents were used fo r immunostaining with anti-PI(4,5)P2 antibody (AM-212) and anti-PI5KI antibody. Upper panel: 4% paraformaldehyde fixation and 0.05% Triton X-100 permeab ilization results in nuclear localization of PI(4,5)P2 (A) and PI5KI (B). Middle panel: 4% para formaldehyde fixation and 10 g/ml digitonin solution permeablization results in patchy distributions of PI(4,5)P2 (E) and PI5KI (F) at the plasma membrane. Lower panel: 4% paraformaldehyde + 0.05% glutaraldedhyde fixation a nd 0.05% Triton X-100 permeabli zation results in leading edge localization of PI(4,5)P2 (I) and PI5KI (J). Note also the intracellular localization of PI5KI (arrow head). Hoechst 33258 stai ns for nucleus (C, G and K). Merged view for each panel is shown in (D,H and L). Scale bar, 5 m. Immunostaining patterns of PI(4,5)P2 and PI5KI with optimized reagents (4% paraformaldehyde+0.05% glutaraldedhyde for fixation and 10 g/ml digitonin solution for permeablization) are shown in Figure 3-6A and 3-6B respectively. Note the clear leading edge co -localization of PI(4,5)P2 and PI5KI (Figure 3-6 C) PI(4,5)P2 PI5KI Hoechst merge 4% PFA 0.05% Triton X-100 4% PFA 10 g/ml Digitonin A B C D E F G H I J K L 4% PFA + 0.05% GTA 0.05% Triton X-100
40 Figure 3-3. Comparison of PI(3,4,5)P3 and PI(4,5)P2 immunostaining. fMLP stimulated HL-60 cells were fixed and permeablized with opt imized reagents (refer to Fig. 2), and immunostained with anti-PI(3,4,5)P3 antibody (A) or anti-PI(4,5)P2 antibody, AM212 (B). Fluorescence intensity profiles, al ong the white line pa ssing through the cell, are also shown for both the antibodies. Note the similar patterns of localizations of PI(3,4,5)P3 and PI(4,5)P2. Scale bars, 5 m. 0 30 60 90 051015 0 30 60 051015 RelativePosition FluorescenceIntensity PI(3,4,5)P3 PI(4,5)P2 PI(3,4,5)P3 PI(4,5)P2 A B
41 Figure 3-4. The localization of PI(4,5)P2 is not an artifact due to membrane folds. Differentiated HL-60 cells were either stained with membrane dye, DiO or immunostained with PI(4,5)P2 antibody, AM-212. Upper panel: Four repr esentative cells of DiO staining in fMLP stimulated HL-60 cells (A,B,C ,D) and the corresponding fluorescence intensity profiles along the periphery (E). Tr end of the intensity profile is shown by thick black line (E). Lower panel: Fo ur representative cells of PI(4,5)P2 antibody staining in fMLP stimulated HL-60 cells (F,G,H,I) a nd the corresponding fluorescence intensity profiles along the periphery (J). Trend of the intensity profile is shown by thick black line (J). Scale bars, 10 m. 00.250.50.751A D C BE A B C D Normalized distance Fluorescence Intensity along periphery Membrane dye, DiO
42 Figure 3-4. Continued F G H I 00.250.50.751F I H GJNormalized distance Fluorescence Intensity along periphery PI(4,5)P2
43 Figure 3-5. PI(3,4,5)P3 and PI5KI colocalize at the leading e dge of fMLP-stimulated HL-60 cells. Differentiated HL-60 cells were stimul ated with fMLP, fixed and permeablized with optimized reagents (refer to Fi g. 2) and then immunostained with antiPI(3,4,5)P3 antibody (A) and anti-PI5KI antibody (B). PI(3,4,5)P3 and PI5KI colocalize at the leading e dge of the HL-60 cells (C). The intensity profiles of PI(3,4,5)P3 and PI5KI along the white line are s hown above the corresponding stains. The data is representative of 24 cel ls analyzed in 3 independent experiments. Scale bar, 5 m. 0 30 60 90 0510 RelativePosition FluorescenceIntensity 0 30 60 90 0510PI(3,4,5)P3 PI5KI A PI(3,4,5)P3 B PI5KI C merge Front Back
44 Figure 3-6. PI(4,5)P2, PI5KI and PI5KI colocalize at the leading edge of fMLP-stimulated HL-60 cells. Differentiated HL-60 cells we re stimulated with fMLP, fixed and permeablized with optimized reagents (4% paraformaldehyde + 0.05% glutaraldedhyde for fixation and 10 g/ml digitonin solution for permeablization). Cells were immunostained w ith either anti-PI(4,5)P2 (A) and anti-PI5KI antibodies or anti-PI(4,5)P2 (D) and anti-PI5KI (E) antibodies. Colocalization of PI(4,5)P2, PI5KI and PI5KI is shown in the merged images (C, F). The fluorescence intensity profiles of PI(4,5)P2, PI5KI and PI5KI along the white line passing through the cell ar e shown above the corresponding stains. PI(4,5)P2 and PI5KI colocalization data is re presentative of 29 cells analyzed in 3 independent experiments. PI(4,5)P2 and PI5KI colocalization data is representative of 26 cells analyzed in 3 independent e xperiments. Scale bar, 5 m. 0 30 60 90 051015 0 30 60 90 051015 RelativePosition FluorescenceIntensit A PI(4,5)P2 B PI5KI C merge PI(4,5)P2 PI5KI Front Back
45 Figure 3-6. Continued 0 30 60 90 051015 0 30 60 90 051015 RelativePosition FluorescenceIntensity D PI ( 4,5 ) P2 E PI5KI F mer g e PI(4,5)P2 PI5KI Front Back
46 Figure 3-7. Effect of PI 3K inhibition on PIP3 and PIP2 polarizations. Differentiated HL-60 cells stimulated with 1 M fMLP show clear leading edge localizations of PIP3 (A) and PIP2 (D). Treatment of dHL-60 cells with 50 mM pan-PI3K inhibitor (LY294002) or 25 mM PI3K specific inhibitor abolishes the le ading localizations of not only PIP3 (B,C respectively) but also PIP2 (E,F respectively).
47 Figure 3-8. Effect of PI 3K inhibition on PIP3 and PI4KII polarizations. Differentiated HL-60 cells stimulated with 1 M fMLP show clear leading e dge localizations of PI4KII (A) and PIP3 (B). Treating these cells with 50 M pan-PI3K inhibitor (LY294002) diminishes the leading local izations of not only PIP3 (E) but also of PI4KII (D). Merged images are also shown (C, F).
48 CHAPTER 4 ACTIN-BINDING PROTEIN, CORONIN-1A IN NEUTROPHIL CHEMOTAXIS Coronin was originally identified as a major membrane-associated protein in the migratory ameboid form of the slime mold Dictyostelium discoideum (de Hostos et al., 1991). The name coronin was chosen because the protein was conc entrated at the crown-like leading edge, or corona, of these migratory cells. Coronin gene knockout resulted in defective cell division and inability of the ameba to migrate toward a source of cyclic AMP, suggesting a role in chemotaxis (de Hostos et al., 1993). Later studies showed that homologues of Dictyostelium coronin are found in most eukaryotes. The coronins be long to theWD (tryptopha nÂ–aspartate) or WD40 family of proteins, the pr ototype of which is the subunit of trimeric G proteins. WD40 proteins generally have a role in mediating specific proteinÂ–protein interactio ns important in many aspects of cellular function (Li and Roberts, 2001 ). Coronins appear to function primarily in association with the membrane cy toskeleton through interactions w ith filamentous actin (F-actin) and the Arp2/3 protein complex, which plays a role in generating branches in the actin filament network (Humphries et al., 2002). The mammalian ge nome contains seven distinct, but related, coronin family members (de Hostos et al., 1993; Rybakin and Clemen, 2005) each of which is an abundant cytoplasmic protei n with cell type-specific expr ession patterns. One mammalian coronin family member, referred to here as coronin-1a, is restrict ed in expression to hematopoietic cells. This protein has been identif ied in different ways by several groups and is consequently also known as coronin 1, p57, Cla bp, clipinA, TACO, or simply coronin (de Hostos, 1999). In macrophages and lymphocytes coronin-1a accumulates at sites of rearrangements of the actin cy toskeleton (Rybakin and Clemen, 2005). Studies looking at the biological role of coronin-1a have implicated coronin-1a in pha gocytosis (Ferrari et al., 1999), Tcell activation (Nal et al., 2004), an d integration of extracellular si gnals to the actin cytoskeleton
49 in leukocytes (Gatfield et al., 2005). Recently, ge ne knockout of coronin-1a in murine T cells leads to reduced ability to res pond to chemotactic gradients and al so increased susceptibility to apoptotic stimuli (Foger et al., 2006). Although the relationship be tween the actin cytoskeleton and coronin-1a is incompletely understood, these properties have lead researchers to believe that coronin-1a is involved in modul ating rearrangement of the actin cytoskeleton during immunespecific functions (Oku et al., 2005). All the above evidence suggests th at coronin-1a might also play a role at the leading edge actin complex formation in neutrophil-like HL 60 cells. Since there were no good coronin-1a antibodies available, first I worked on the de velopment and characteri zation of coronin-1a antibody. We were able to generate a highly sp ecific rabbit coronin-1a antibody. Immunostaining in chemotactic HL60 cells showed a leading edge localization of coronin-1a, which was associated with actin as reveal ed by costaining with phalloidin. Generation of Coronin-1a Antibody A cDNA encoding human coronin-1a was isol ated by PCR from a human leukocyte large insert cDNA library (Clontech; Mountain View, CA) using primers designed to amplify the fulllength coding sequence. A band of the expected size was isolated and ligated into PCR2.1 T overhang shuttle vector, and the sequence was ve rified by direct sequencing. The insert was found to be 100% identical to the human coronin-1a sequence (Genbank accession NM_007074; Figure 4-1). The full-length open-reading frame of the sequence was further subjected to PCR to add a 5Â’ EcoRI site immediately in front of the initiator methionine codon and a 3Â’ SalI site immediately after the stop codon. Following primers were used: Forward primer: 5Â’-GAATTCATGAGCCGGCAGGTGGTCCG-3Â’ Reverse primers: 5Â’-GTCGA CGCGGGGCTCTACTTGGCCTGG-3Â’
50 The PCR product was then ligated into pATH11 vector (Figure 4-2) for expression of the full-length protein in Escherichia coli. A Tr p-E-coronin-1a fusion protein was generated essentially as described previ ously (Harris et al., 1991). Brie fly, fusion protein expression was induced by indoleacrylic acid, and th e fusion protein was enriched by subjecting the bacteria to an inclusion-body preparation. Th is inclusion-body material wa s dissolved in 6M urea and further purified by preparative SDS-PAGE. Th e purified Trp-E-coroni n-1a construct was injected into rabbits to ge nerate a polyclonal antibody. Characterization and Immunostai ning of Coronin-1a Antibody Because coronin-1a is found in the cells of he matopoietic lineage, we expected to find it in neutrophils. Therefore we grew cultures of HL-60 cells, a human cell line that can be differentiated to a neutrophil morphology. Cells were differentiated by treatment with DMSO, and addition of the peptide fMLP further activ ated a migratory and chemotatic morphology. HL60 cells in suspension were stimulated with 100 nM formyl-Met-Leu-Phe (fMLP) for 2 min. Whole cell lysate from these cells was used in western blotting (see Appendi x A) to characterize rabbit coronin-1a antibody. A single band at ~ 57 kDa was observed, the expected molecular mass for coronin-1a (Figure 4-3, A). We also i mmunostained HL60 cells w ith rabbit coronin-1a antibody and found that it is associat ed with actin at th e leading edge, as seen by costaining with fluorescent phalloidin (Figure 4-3, B-D)
51 1 cattgtcttg acaagagcat cttcagcggg cgagtccccg gctcctccag ctccttcctc 61 ctcttcctcc tcctcctcca cctccggctt ttgggggatc actgtcctct ctcggcagca 121 gaatgagccg gcaggtggtc cgctccagca agttccgcca cgtgtttgga cagccggcca 181 aggccgacca gtgctatgaa gatgtgcgcg tctcacagac cacctgggac agtggcttct 241 gtgctgtcaa ccctaagttt gtggccctga tctgtgaggc cagcggggga ggggccttcc 301 tggtgctgcc cctgggcaag actggacgtg tggacaagaa tgcgcccacg gtctgtggcc 361 acacagcccc tgtgctagac atcgcctggt gcccgcacaa tgacaacgtc attgccagtg 421 gctccgagga ctgcacagtc atggtgtggg aaatcccgga tgggggcctg atgctgcccc 481 tgcgggaacc cgtcgtcacc ctggagggcc acaccaagcg tgtgggcatt gtggcctggc 541 acaccacagc ccagaacgtg ctgctcagtg caggttgtga caacgtgatc atggtgtggg 601 acgtgggcac tggggcggcc atgctgacac tgggcccaga ggtgcaccca gacacgatct 661 acagtgtgga ctggagccga gatggaggcc tcatttgtac ctcctgccgt gacaagcgcg 721 tgcgcatcat cgagccccgc aaaggcactg tcgtagctga gaaggaccgt ccccacgagg 781 ggacccggcc cgtgcgtgca gtgttcgtgt cggaggggaa gatcctgacc acgggcttca 841 gccgcatgag tgagcggcag gtggcgctgt gggacacaaa gcacctggag gagccgctgt 901 ccctgcagga gctggacacc agcagcggtg tcctgctgcc cttctttgac cctgacacca 961 acatcgtcta cctctgtggc aagggtgaca gctcaatccg gtactttgag atcacttccg 1021 aggccccttt cctgcactat ctctccatgt tcagttccaa ggagtcccag cggggcatgg 1081 gctacatgcc caaacgtggc ctggaggtga acaagtgtga gatcgccagg ttctacaagc 1141 tgcacgagcg gaggtgtgag cccattgcca tgacagtgcc tcgaaagtcg gacctgttcc 1201 aggaggacct gtacccaccc accgcagggc ccgaccctgc cctcacggct gaggagtggc 1261 tggggggtcg ggatgctggg cccctcctca tctccctcaa ggatggctac gtacccccaa 1321 agagccggga gctgagggtc aaccggggcc tggacaccgg gcgcaggagg gcagcaccag 1381 aggccagtgg cactcccagc tcggatgccg tgtctcggct ggaggaggag atgcggaagc 1441 tccaggccac ggtgcaggag ctccagaagc gcttggacag gctggaggag acagtccagg 1501 ccaagtagag ccccgcaggg cctccagcag ggtcagccat tcacacccat ccactcacct 1561 cccattccca gccacatggc agagaaaaaa atcataataa aatggcttta ttttctggta Figure 4-1. Nucleotide sequence of homo sapi ens coronin 1A (CORO1 A), (Genbank accession NM_007074). Start and stop codons have been marked with gray background.
52 Figure 4-2. The pATH11 vector showing trpE ge ne. Coronin-1a gene was inserted between EcoRI and SalI restriction sites in the multiple cloning site (MCS).
53 Figure 4-3. Characterization a nd immunostaining of rabbit co ronin-1a antibody. An immunoblot of differentiated HL60 cell lysate staine d with the coronin1a antibody (A). The numbers indicate appropriate molecular ma ss standards. A prominent band at ~57 kDa is as expected for coronin-1a. Imag es of differentiated and fMLP-stimulated HL60 cells stained with antibody to coronin1a (green channel in B,D), fluorescent phalloidin (red channel in C,D), and DAP I DNA fluorescent intercalating reagent (blue in D). Coronin-1a is membrane associ ated and concentrated at the leading edge (top left) of this actively chemotactic cell. A B C D
54 CHAPTER 5 CONCLUSIONS Localization of PI(4,5)P2 at the Leading Edge A common theme in the literature is the re gulation of various pr ocesses by PI(4,5)P2 levels. Yet, whole-cell analyses sh ow only modest changes in PI(4,5)P2 levels. In the particular case of neutrophils, exposure to fMLP causes the whole-cell PI(4,5)P2 levels to decrease from 5 to 3.5 mM before their subsequent recovery to pr e-stimulus levels (Stephens et al., 1993). This has led to the suggestion that these processe s are regulated by local changes in PI(4,5)P2 levels. However, the localization of PI(4,5)P2 remains the subject of considerable debate. The controversy stems from two main issues. First, there are concerns rega rding the specificity of PH-PLC as a marker for PI(4,5)P2. Early studies showed that in unstimulat ed rat basophilic leukemia cells, PH-PLC was bound to the membrane, and stimulation with agoni sts led to the rele ase of GFP-PH-PLC into the cytosol (Stauffer et al., 1998). It was c oncluded that this reflected th e PLC-mediated hydrolysis of PI(4,5)P2. However, Hirose et al found that that GFP-PH-PLC binds to Ins(1,4,5)P3 with an affinity that is 20-fold higher than its affinity for PI(4,5)P2 (Hirose et al., 1999). Given this fact, it is not clear whether the migration of PH-PLC from the membrane to the cytosol reflects a decrease in PI(4,5)P2 levels or an increase in the cytosolic I(1,4,5)P3 levels. Based on the analysis of a kinetic model, Xu et al have argue d that the answer depends on the concentration of GFP-PH-PLC in the cell (Xu et al., 2003). Thus, it can be difficult to interpret the results obtained from experime nts involving PH-PLC as a marker for PI(4,5)P2. Second, the observed loca lization of PI(4,5)P2 can be an artifact resulting from membrane folding. Tall et al studied the spatiotemporal dyna mics of PH-PLC in NIH-3T3 fibroblasts (Tall et al., 2000). They observe d colocalization of PH-PLC and actin in highly dynamic structures
55 which were identified as ruffles. However, va n Rheenen & Jalink showed subsequently that these ruffle-like structures were in fact memb rane-rich microdomains (van Rheenen and Jalink, 2002). In this work, we used immunostainin g to study the localiz ation of PI(4,5)P2 and PI5 kinases. We found that a slight modification of the standard fixation and permeabilization protocols reveals sharp gradients of PI(4,5)P2 despite its high basal leve ls, and these gradients are not due to membrane enrichment. Furthermore, these modified protocols also reveal the colocalization of PI(4,5)P2 and PI5-kinases much more clearl y than the standard immunostaining protocols. Indeed, Doughman et al used th e standard immunostaining protocols (4% PFA fixation for 10 min, followed by permeabilizatio n with 0.2% Triton X-100 for 10 min) to investigate the coloca lization of PI(4,5)P2 and PI5KI at the membrane ruffles of PDGFstimulated fibroblasts (Doughman et al., 2003). Co mparison of Figure 3-6 of this work with Figures 2C of (Doughman et al., 2003) shows that the colocalization is much clearer with the modified protocol. The localization of PI(4,5)P2 was also observed by Watt et al (Watt et al., 2002), who circumvented the problems associ ated with transfection of PH-PLC by using an on-section labeling approach in which ultrathin thaw ed cryosections were incubated with PLC -PH-GST, followed by antibodies against GST and Protein A-gold. The labeling was found to be markedly concentrated in the lamellipodium. Positive Feedback Loop Mechanism for PI(3,4,5)P3 Polarization The data in the literature s uggests that all three positive f eedback pathways 1-3 operate (Figure 2-4). This evidence is based on the fo llowing two observations. First, in response to chemoattractant stimulation, the PI P kinases associated all three pathways ar e recruited to the
56 leading edge. Second, Rac, which is a critical com ponent of all three pathways, interacts with or influences the PIP kinases of all three pathways (Srinivasan et al., 2003). Indeed, the existence of pathway 1 is supported by following facts. Rac-GTP binds to Type IA PI3K, a nd activates it (Bokoch et al., 1996). Inhibition of PI3K a Type IA PI3K, results in decreased PI(3,4,5)P3 levels, and impairs the gradient sensing mechanism (Sadhu et al., 2003). There is evidence supporting the exis tence of pathway 2 in fibroblasts. Rac-GDP and Rac-GTP bind to PI 5KI both in vitro and in vivo. Upon chemoattractant stimulation, PI5KI translocates from the golgi to the plasma membrane, where it stimulates membrane ruffling and PI(4,5)P2 synthesis (Doughman et al., 2003). Finally, Wei et al have obtained the following ev idence supporting the existence of pathway 3 in HeLa cells (Wei et al., 2002). Upon PDGF stimulation, PI4KII but not PI4KII translocates from the cytosol to membrane ruffles in the plasma membrane. Overexpression of constitutively active R acV12 induces membrane ruffling, and PI4KII translocation, whereas dominant-n egative RacN17 blocks PI4KII translocation. We studied the spatial distribution of PI(4,5)P2, PI(3,4,5)P3, PI5KI and PI5KI in neutrophil-like HL-60 cells e xposed to uniform fMLP concentrations. We found the following results. Not only PI(3,4,5)P3, but also PI(4,5)P2, localize at the leading edge of the cell, and the fluorescent intensity gradient of PI(4,5)P2 is comparable to that of PI(3,4,5)P3. The enzymes, PI5K and PI5K which catalyze the synthesis of PI(4,5)P2, colocalize with PI(4,5)P2. The polarization of not only PI(3,4,5)P3, but also PI(4,5)P2, PI5K and PI5K is diminished by LY294002, an pan-PI 3K inhibitor or by IC87114, PI3K inhibitor. The enzyme, PI4KII colocalizes with PI(3,4,5)P3 at the leading edge and the inhibition of PI3K leads to diminished PI4KII and PI(3,4,5)P3 polarizations.
57 These results imply that the complementary regulation of the PI3K-PTEN couple and the positive feedback due to Rac-mediated PI3K activ ation (loop 1 in Figure 24) cannot be the sole or dominant mechanisms of PI(3,4,5)P3 polarization, since both mech anisms imply depletion, or at best, no change, of the PI(4,5)P2 levels at the leading edge The localization of PI(4,5)P2 implies that some pathway stimulating PI(4,5)P2 synthesis is also activated in response to chemoattractant stimulation. The localization of PI5KI and PI5KI at the leading edge suggests that one such pathway is a PI5KI-media ted positive feedback loop from PI(3,4,5)P3 to PI(4,5)P2. PI3K inhibition experiments show that this pathway is PI(3,4,5)P3 dependent. PI4KII experiments show that there is a possibility of an other PI(3,4,5)P3-dependent positive feedback loop (loop 3 in Figure 2-4). The phosphatase PTEN certainly plays an important role in PI(3,4,5)P3 polarization. In Dictyostelium PTEN-null mutants exposed to cAMP develop a broad PI(3,4,5)P3 localization (Funamoto et al., 2002; Iijima and Devreotes, 2002). In neutrophils, suppression of PTEN expression with PTEN-specific siRNA results in pronounced elevation of p-Akt activity, a sensitive and specific marker for PI(3,4,5)P3 levels (Li et al., 2005). Yet, the localization of PI(4,5)P2 at the leading edge suggests that the role of PTEN is mo re complex than that implied by the model based on complementary regulation of PI3K and PTEN.
58 APPENDIX MATERIALS AND METHODS Materials Paraformaldehyde (reagent grade), glutaraldehyd e (50% solution in wa ter, reagent grade) and Triton X-100 were from Fisher Scientific (Suwanee, GA); digitonin, fMLP and RPMI-1640 medium were from Sigma-Aldrich (St. Loui s, MO). Mouse monoclonal anti-PtdIns(3,4,5)P3 IgG and PIP strips were from Eche lon Biosciences (Salt Lake Cit y, UT), rabbit peptide antibodies, anti-PIP5KI and anti-PIP5KI pan, were gifts from R. Anderson (Wisconsin) and P. De Camilli (Yale University, New Haven, CT), resp ectively. Monoclonal anti-PtdIns(4,5)P2 antibodies, clone KT-10 (21) and clone AM-212 (22) were kindly provided by K. Fukami (University of Tokyo, Japan) and M. Umeda (ICR, Kyoto Univer sity, Japan). Membrane dyes (DiO vibrant, DiI), secondary antibodies, Alexa Fluor 488 (g reen fluorescent) and Alexa Fluor 594 (redfluorescent) goat anti-mouse or rabbit, and nuclear stain (Hoechst 33258) were from Invitrogen (Carlsbad, CA). Cell Culture and Differentiation Culture and differentiation of HL-60 cells were performed as described (6). Briefly, cells were grown in RPMI-1640 culture media supp lemented with 10%FBS, 1% AmphotericinB and 1% pen-strep/glutamine in a humid chamber at 37C with 5% CO2. In a T-75 flask, cells reach a density of 1.0-1.5 x 106 cells/ml in 3-4 days. For differentiation, 4 x 106 cells were seeded in 19.75 ml (final volume) supplemented culture media and then 0.25 ml (1.25%) DMSO was added. The cells were propagated for 6 days without changing the medium. Dot Blot Assay The PIP strip was blocked with 3% BSA in TBS overnight at 4 C, and incubated with primary antibody for 1 hour. After extensive wa shing in TBS containing 0.05% Tween 20, the
59 strip was incubated with horse radish peroxidase-conjugated se condary antibody for 1 hour, and then visualized with the enhanced chem iluminescence kit (Amersham Biosciences). Enzyme-Linked Immunosorbent Assay (ELISA) The wells of the high-binding polystyrene microtiter plates (Thermo Scientific) were coated with various phospholipids (1 ug/well) in et hanol. Plates were left at room temperature (RT) until complete ethanol evaporation. The we lls were blocked with 3% BSA solution in TBS for 1 hour at RT. Serial dilution of AM-212 seru m in 3% BSA was applied to each phospholipid row for 1 hour at RT. After washing four times in 0.05% TBS-tween, alkaline phosphatase conjugated goat anti-mouse IgG (Sigma) was applie d at a dilution of 1:2000, for 1 hour at RT. Wells were washed four times in 0.05% TBS-tween, 100 l of pNPP substrate solution (Sigma) was added, and absorbance was measured at 405 nm. Chemotaxis Assay Differentiated HL-60 cells were wash ed once in RPMI-1640/25mM HEPES and resuspended at a concentration of 3.0*106 cells/ml in modified HBSS (mHBSS) containing 150 mM NaCl, 4mM KCl, 1.2 mM MgCl2, 10 mg/ml gl ucose, and 20 mM HEPES, pH 7.2. The cells (3*105 in 100 l) were plated on the fibronectin coated (0.05 mg/ml) sterile no.1 coverslips, rimmed with a square agarose spacer 10 mm in length and 1 mm in height. Cells were then incubated in a humid chamber at 37C with 5% CO2 for 15-20 min, and non-adherent cells were removed by two washes in mHBSS. Immunostaining and Microscopy The cells were stimulated with 1M fMLP for 2 minutes and then fixed for 10 min at room temperature in different fixative solutions as described in resu lts section. The cells were then permeabilized and blocked for 30 min in a detergent solution (see results section) containing 5% goat serum, followed by overnight incubation with appropriate primary antibodies at 40C. After
60 three successive washes in TBS, the cells were incubated with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594 goat anti-mouse or rabbit) for 4-5 hours, washed three times in TBS, and mounted on a glass slide. Fluorescence mi croscopy was done on a deconvolution microscope (DeltaVision RT from Applied Precision) and fo llowing filter sets were us ed, FITC (excitation: 490/20 nm, emission: 528/38), RD-TR-PE (excit ation: 555/28 nm, emission: 617/73), Cy5 (excitation: 640/20 nm, emission: 685/40), and DAPI (excitati on: 360/40 nm, emission: 457/50). No bleed-through of fluorescence signal was obser ved from one channel to another channel. Images were analyzed using softWoRx (App lied precision) and ImageJ (NIH) softwares. Plasma Membrane Labeling Differentiated HL-60 cells were resuspe nded in RPMI-1640/25 mM HEPES. 5ul DiO Vybrant or DiI was added and cells were incubated at 370C for 5-7 mins. Cells were washed once with RPMI-1640/25 mM HEPES to remove the excess dye, resuspended in mHBSS and then plated on fibronectin coated coverslips for the chemotaxis assay. Western Blotting Differentiated HL-60 cells in suspension we re stimulated with 100 nM formyl-Met-LeuPhe (fMLP) for 2 min. Whole cell protein was precipitated by trichl oroacetic acid (TCA) treatment and then dissolved in LaemmliÂ’s SD S buffer by boiling/vortexing for 4 min. Lysates were separated with 7.5% SDS-PAGE and transfe rred to PVDF, blocked in 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 hr, and in cubated with 1:10,000 rabbi t coronin-1a antisera overnight at 4C. The PVDF membrane was wash ed three times in 0.05% Tween-20 in TBS and then incubated with 1:10,000 horseradish peroxi dase-conjugated anti-rabbit antibody (Amersham Biosciences; Pittsburgh, PA) for 1 hr at room te mperature. After further washing in TBS plus Tween-20, the signal was detected with reagen ts in the AmershamÂ’s chemiluminescence kit according to the manufacturerÂ’s instructions.
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