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Vol. 14, Issue 12, 5028-5037, December 2003
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Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205
Submitted May 28, 2003;
Revised October 7, 2003;
Accepted October 10, 2003
Monitoring Editor: Mary Beckerle
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Schmidt and Hall, 1998; Higgs and Pollard, 2001; Pollard and Borisy, 2003).
Many of the events underlying chemotaxis have been elucidated in the social amoebae Dictyostelium discoideum and in neutrophils (Zigmond, 1989; Parent and Devreotes, 1999; Rickert et al., 2000; Chung et al., 2001; Heit et al., 2002; Wang et al., 2002). Studies of these cells suggest that directional sensing is established by phosphatidyl-3',4',5'-triphosphate and/or phosphatidyl-3',4'-bisphosphate [PI(3,4,5)P3/PI(3,4)P2] production and degradation at the plasma membrane in response to G-protein signaling. The levels of PI(3,4,5)P3 are regulated by PI 3-kinases (PI3Ks) and the PI 3-phosphatase, PTEN (Buczynski et al., 1997; Hirsch et al., 2000; Li et al., 2000; Sasaki et al., 2000; Funamato et al., 2001, 2002; Iijima and Devreotes, 2002; Stocker et al., 2002). In resting D. discoideum cells, PI3Ks are found in the cytosol, whereas a fraction of PTEN is localized at the cell membrane. When cells are uniformly exposed to chemoattractant, the PI3Ks rapidly bind to the membrane and PTEN dissociates. As stimulation continues, the PI3Ks return to the cytosol, and PTEN reassociates with the membrane. When cells are exposed to a gradient of chemoattractant, PI3Ks and PTEN bind to the membrane at the front and rear of the cell, respectively. This leads to the selective accumulation of PI(3,4,5)P3/PI(3,4)P2 at the leading edge. Studies of pten- cells demonstrate a causal relationship between PI(3,4,5)P3/PI(3,4)P2 levels and actin polymerization. When exposed to a uniform stimulus, pten- cells exhibit a prolonged and stronger association of PH domain with the cell membrane and a parallel increase in actin polymerization. Placed in a chemoattractant gradient, pten- cells fail to produce sharp localizations of PI(3,4,5)P3/PI(3,4)P2 and F-actin at the cell's leading edge and chemotaxis is severely impaired (Funamato et al., 2001; Iijima and Devreotes, 2002).
Although PI(3,4,5)P3/PI(3,4)P2 clearly regulates actin polymerization, polarity, and directional sensing, its essential function has been difficult to establish. Disruption of PI3K activity (deletion of two of three D. discoideum PI3Ks) impairs but does not block the ability of cells to sense and orient in chemotactic gradients (Buczynski et al., 1997; Funamoto et al., 2001). Similarly, disruption of mammalian PI3Ks and PTEN interfere with chemotaxis and motility, but the underlying biochemical mechanisms have not been defined. Also, PI3K inhibitors, LY294002 and wortmannin, have been reported to inhibit but do not prevent chemotaxis in a variety of cell types, unless unphysiologically high concentrations are used (Wu et al., 2000; Wymann et al., 2000; Wang et al., 2002; unpublished observations). Moreover, mutations or inhibitors of PI3K do not significantly affect the rapid phase of chemoattractant-induced actin polymerization (Li et al., 2000; Funamato et al., 2001).
Previous studies analyzing actin polymerization in D. discoideum and neutrophils suggested the existence of a biphasic response to a uniform stimulus (Condeelis et al., 1988; Norgauer et al., 1994; Postma et al., 2003). The cells undergo a series of discrete cell shape changes during the two phases of actin polymerization (Futrelle et al., 1982; Condeelis et al., 1990; Caterina and Devreotes, 1991; Chen et al., 1996). During the first peak, cells freeze motion; then at 
30 s, as F-actin levels drop precipitously, cells round up or "cringe." During the second phase of actin polymerization, which lasts several minutes, the cells extend new pseudopods from multiple regions and start to move. Cells return to normal morphology shortly thereafter. Our data here shows that in D. discoideum the two phases of actin polymerization closely parallel a biphasic accumulation of PI(3,4,5)P3/PI(3,4)P2 and that the second phase of actin polymerization depends directly on the slow phase of PI(3,4,5)P3 accumulation. We explore the role that each component of the biphasic responses has in directional sensing and during chemotaxis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Cell lines used included wild-type cells (WT: AX2 or AX3); SCAR-GFP (from Dr. Saxe) expressed in AX2; PHCRAC-GFP expressed in AX3; PHCRAC-RFP and Coronin-GFP (from Dr. Gerisch) expressed in AX3; and pi3k1-/pi3k2- (pi3k1-/2-, from Dr. Firtel), pten-, and PHCRAC-GFP expressed in pten-.
Actin Polymerization Assay
Actin polymerization assays were carried out as previously described (Iijima and Devreotes, 2002; Zigmond et al., 1997). To achieve a basal level, developed cells were shaken at 200 rpm with 2 mM caffeine in PM buffer for 30 min, collected, washed, resuspended at 3 x 107/ml with 2 mM caffeine in PM buffer (10 mM phosphate buffer, 2 mM MgSO4), and shaken at room temperature for no more than 30 min. At various time points after adding 1 µM cAMP, aliquots of cells were taken, fixed, and stained with TRITC-phalloidin. (Buffer for fix and stain: 3.7% formaldehyde, 0.1% Triton, 250 nM TRITCPhalloidin, 20 mM KPO4, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8). F-actin was proportional to the amount of phalloidin fluorescence (Excitation wavelength: 540 nm. Emission wavelength: 570 nm.) extracted from cell pellets.
Immunoblot Analysis of PH Domain Translocation
The assay was performed as described (Parent et al., 1998). Cells were pretreated with caffeine, then washed, and resuspended in PM buffer at 8 x 107/ml. At indicated time points after stimulation with 1 µM cAMP, fractions of cells were filter-lysed into ice-cold PM buffer to terminate the reaction. Membrane fractions were collected and assayed by immunoblot of PHCRAC-GFP with anti-GFP antibody.
Fluorescence Microscope
Images of living cells were observed using Zeiss inverted microscopes (Axiovert 135TV and Axiovert 100; Zeiss, Oberkochen, Germany) as previous described (Parent et al., 1998). Cells were allowed to adhere on a chamber (Lab-tek, Nalge Nunc, Rochester, NY) and stimulated by 1 µM cAMP. To image double-labeled cells, filter wheels carrying the exciters HQ480/25 and D565/25 were used along with the emitters HQ525/40 and D620/60 (filters from Chroma, Rockingham, VT). IP lab and Image J (Scanalytics, Fairfax, VA) software packages were used to collect and process data.
Chemotaxis Assay and Quantification
Chemotactic movements of cells to a micropipette containing cAMP were performed as described (Parent et al., 1998). Cells were allowed to adhere on a chambered coverglass (Lab-tek, Nalge Nunc). A micropipette filled with 10 µM cAMP was positioned, and images of moving cells were recorded. IP lab and Image J software packages were used to collect and process data.
Speed of chemotaxis was calculated from the distance a cell covered divided by the time. Chemotactic Index was defined as the cosine of the angle formed by the line between the cell start point and the micropipette tip point and the line from the cell start point to the cell ending point (Iijima and Devreotes, 2002). Speed and Chemotactic index were measured during a period of 200 s.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Huang et al., 2003; Lemmon, 2003). To assess actin dynamics in living cells, we followed the movement of coronin-GFP to the cortex. Coronin is known to localize with newly formed F-actin and has been suggested to bind to the Arp2/3 complex (de Hostos, 1999; Mishima and Nishida, 1999; Humphries et al., 2002). In response to a uniform stimulus, both proteins displayed two phases of association to the cell perimeter. Within seconds, PHCRAC-RFP was recruited uniformly to the cell membrane. It then returned to the cytosol and a second phase, localized to membrane extensions, ensued (Figure 1B, red). The two phases of PHCRAC translocation have also been observed by Postma et al. (personal communication). For coronin-GFP, the first translocation was uniformly localized at the cell cortex, whereas the second was localized to regions of newly formed pseudopods (Figure 1B, green). The spatial and temporal features of each response were similar, but the PHCRAC-RFP recruitment to the membrane appeared to precede association of coronin-GFP with the cortex. However, increased temporal resolution will be required to verify this point. The PHCRAC-RFP clearly localized more peripherally than coronin-GFP (Figure 1B, merged). We observed similar biphasic membrane recruitments with another actin-binding protein, SCAR (from Dr. Saxe, Bear et al., 1998) and also with PI3K2 (unpublished data).
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We examined the biphasic response in WT and pten- cells in the absence or presence of the PI3K inhibitor LY294002 (LY). PHCRAC-GFP was used as a marker for PI(3,4,5)P3/PI(3,4)P2 accumulation. As previously reported, levels of PI(3,4,5)P3/PI(3,4)P2 production and actin polymerization were significantly larger during the period from 30 to 180 s in the pten- cells (Figure 2, A and B). The first peak recruited PHCRAC-GFP uniformly to the cell perimeter. During the large second phase in the pten- cells, PHCRAC-GFP bound to the membrane at the points where cells were spreading. Addition of 30 µM LY caused a rounding and a reduction of the membrane ruffles in both WT cells and pten- cells (Figure 2A). When these treated cells were stimulated, the second phase of PHCRAC-GFP translocation was selectively inhibited. In both cell types treated with LY, the first peak of chemoattractant-induced PHCRAC-GFP translocation was still detectable by microscopy and by immunoblot (Figure 2B).
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With these observations in hand, we reexamined actin polymerization using PI3K inhibitors as well as pi3k1-/2- and pten- cells. In WT cells, 30 µM LY blocked the second phase of actin polymerization with little effect on the initial rapid phase (Figure 3A). In pten- cells, the enlarged second phase of actin polymerization was selectively inhibited with 30 µM LY or 10 µM wortmannin, another PI3K inhibitor (Figure 3B and unpublished data). Inhibition of the first and second phases of actin polymerization by 30 µM LY in pten- cells were 0 and >85%, respectively. In the pi3k1-/2- cells, there was no obvious second phase and the first phase was insensitive to 30 µM LY (Figure 3C). Higher concentrations of LY than those used in Figure 2 (up to 200 µM) did eventually inhibit the rapid phase of PI(3,4,5)P3/PI(3,4)P2 production. Curiously, these high concentrations, which are likely unphysiological, elevated basal F-actin levels to those normally found at the peak of the rapid phase. Under these conditions, there was little further increase induced by chemoattractant (unpublished data).
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We next examined directed movements of WT and pten- cells treated with 30 µM LY. Addition of the drug, which blocked the second phase of the response to a uniform stimulus, impaired chemotaxis of WT cells. In the absence of LY, the cells moved randomly until exposed to a gradient and then moved toward the micropipette by turning or creating new leading edges. With LY treatment, random movement and membrane ruffles were significantly reduced. When the gradient was applied, the cells polarized and moved toward the chemoattractant, although they moved more slowly and displayed fewer lateral pseudopods. PHCRAC-GFP was detected less often along the leading edge of the treated cells compared with WT cells (Figure 4, top). As previously described, most pten- cells are defective in speed and directional sensing, although there is a subset of pten- cells that displays nearly normal chemotactic indices. Interestingly, although LY impaired chemotaxis of the WT cells, it actually improved the chemotactic responses of pten- cells (Figure 4, bottom). The PHCRAC-GFP staining was more localized, and the cells acquired a narrower leading edge, extended fewer lateral pseudopods, and moved rapidly toward chemoattractant (Figure 4, bottom). For each experimental condition, we quantified the speed and chemotactic index from three sets of independent experiments (Figure 4). Although LY treatment reduced the average speed of WT cells 20%, it increased that of the pten- cells by about twofold. Also, the drug decreased the chemotactic index of WT cells but increased that of the pten- cells. Thus, the second phases of the PI(3,4,5)P3 accumulation and actin polymerization are tightly regulated by PI3K, and optimal levels are required for efficient directed movement. However, cells substantially lacking the second phases or with elevated second phases were still able, albeit inefficiently, to chemotax.
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The two phases of PI(3,4,5)P3 accumulation and actin polymerization were next examined at different stages of development. Under starvation conditions, D. discoideum cells undergo differentiation and become polarized. Growth phase cells are unpolarized and project many lateral pseudopods. As cells differentiate, polarity increases; 7-h stage cells are elongated and maintain a persistent leading edge (Figure 5A). Cells grown in the presence of bacteria, which are quite unpolarized, displayed a second phase of actin polymerization when stimulated with folic acid (unpublished data). In 5-h stage cells, which are relatively unpolarized, we also observed a significant second peak of PI(3,4,5)P3/PI(3,4)P2 and actin polymerization (Figures 1, 2, and 5B). In 7-h polarized cells, the second phases of cell spreading and PHCRAC-GFP translocation were less apparent by microscopy. Similarly, when we examined actin polymerization in 7-h cells, the second phase was significantly reduced (Figure 5).
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Careful examination suggested that the second phase of the response determined the cell's capacity to extend lateral pseudopods in the new direction when the gradient was switched. In 5-h stage cells the leading edge is less defined and is not persistently maintained during movement. By comparison, the 7-h stage cells are polarized and normally turn when changing directions, as occurs when a micropipette containing cAMP is moved to a new location. To quantitate these behaviors, we repeatedly moved a micropipette tip and recorded the time (referred to as "delay time") for the leading edge of the cell to display sustained movement in the new direction. Figure 6A shows examples of this behavior, and data from many events are shown in Figure 6B. For the 5-h cells, the average "delay time" was 48 s and 90% of the events occurred within 1 min. LY treatment of these cells increased the average "delay time" to 146 s. This was similar to that of 7-h cells, where the average "delay time" was 162 s. Interestingly, the pten- cells had a short "delay time" (average 37 s) and often extended lateral pseudopods spontaneously. Treatment of the pten- cells with 30 µM LY increased the "delay time" (average 114 s) toward the WT cell level (Figure 6B). Although the "delay times" of the 5-h cells and pten- cells treated with LY were both similar to the 7-h cells, the treated pten- cells displayed a more normal morphology and behavior (unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Local accumulations of PI(3,4,5)P3 appear to control many actin based cellular extensions including the spontaneous ruffles that occur during random migration as well as the second phase of the chemoattractant-induced response. LY-treated cells display very few membrane ruffles, and pseudopods are mainly distributed at the side of the cell facing the high concentration of chemoattractant. When direction of the gradient is switched, the treated cells display some of the characteristics of more polarized cells. For example, the "delay time" increases substantially upon a gradient switch. It approaches the longer "delay time" of the 7-h cells, which must turn to seek the new gradient. The "delay time" is shorter in 5-h cells because they are able to extend lateral pseudopods and create a new front. Furthermore, pten- cells, which have a very large second phase, are unpolarized, switch fronts rapidly, and have increased spontaneous ruffle activity.
It is unclear why an optimal level of second phase response, which allows cells to rapidly react to a switch in gradient direction, improves the chemotactic response to a stable gradient. We speculate that cells that are unable to react rapidly to gradient shifts may be slow to "correct" their direction if they get "off tract." This deficiency might lead to inefficient directional migration. On the other hand, excess second phase response as occurs in pten- cells also leads to inefficient chemotaxis. In growing cells, which must track motile bacteria and 5-h stage cells, which have not yet been recruited to a specific group of streaming cells, the ability to switch direction easily is advantageous. The second phase of the actin response decreases as cell polarity increases, and most cells have already formed streams during aggregation. This may allow cells to move more rapidly when switching direction is less critical.
The significance of the two phases of actin polymerization has not been fully appreciated, perhaps because the phenomenon has not been consistently observed. In fact, it is difficult to observe unless conditions are carefully controlled. Even in the absence of a stimulus, cells move randomly, making pseudopods and ruffles that contribute to the basal level of filamentous actin. With a uniform stimulus, cells respond synchronously during the first peak, but individual cells generate new extensions and ruffles asynchronously during the second phase (Figures 1B and 2A). These tend to average out and lower the apparent level of the response. In 7-h developed cells, the frequency or intensity of ruffling during the second phase never exceeds that present before the stimulus and thus the second peak of actin polymerization is undetectable.
We are puzzled that the first phase of PI(3,4,5)P3 accumulation is relatively insensitive to PI3K inhibitors. One might speculate that transient loss of PTEN from the membrane contributes to the first phase of PI(3,4,5)P3 production. That is, even when PI3K is quite inhibited, the chemoattractant-induced decrease of PTEN activity is significant enough to elevate PI(3,4,5)P3 levels. However, we ruled out this possibility, because LY treatment does not inhibit the first phase even in pten- cells. The fact that LY inhibits the second peak also indicates that the drug enters the cell efficiently. Once again, it is not clear why the first peak is not inhibited. It is possible that there is an inhibitor-resistant PI3K activity or it may be the drug cannot access the PI3K during the initial phase.
Our results clearly show that the second phase of actin polymerization is controlled directly by the production of PI(3,4,5)P3. But the question remain as to whether PI(3,4,5)P3 is essential for the first peak of actin polymerization. In our experiments, the LY concentration used (30 µM or 50 µM) did not fully inhibit the first phase of PHCRAC-GFP translocation. Therefore, we cannot conclude whether PI(3,4,5)P3 is required for this rapid burst of actin polymerization. Unphysiologically high drug concentrations did substantially decrease the first phase of PI(3,4,5)P3 accumulation. However, the first peak of actin polymerization was impossible to assess because basal levels of F-actin increased substantially. Interestingly, the first peak of actin polymerization was also unaffected in pi3k1-/2- cells, which retain only 10-15% of the WT level of chemoattractant-induced PI(3,4,5)P3 accumulation (Huang et al., 2003). It may be that very few PI(3,4,5)P3 molecules can trigger actin polymerization and there are further amplification steps downstream of PI(3,4,5)P3 production.
Alternatively, a redundant pathway downstream of the G-proteins may control the first peak of actin polymerization. We are currently exploring the conditions that can fully block the rapid response to chemoattractant. It has been suggested that PLC activation and a consequent decrease in PI(4,5)P2 could contribute to the first phase of actin polymerization in mammary carcinoma cells stimulated with EGF (Condeelis, personal communication). We tested this mechanism in D. discoideum by monitoring the chemoattractant-induced actin response in plc- cells (from Dr. Van Haastert) treated with LY. The first peak of the actin polymerization was unaffected under these conditions. Current efforts are focused on other possible intermediates that may directly contribute to the first phase of actin polymerization.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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* Corresponding author. E-mail address: pnd{at}jhmi.edu.
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