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Article |
Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing
Correspondence to Tian Jin: tjin{at}niaid.nih.gov
Gprotein–coupled receptor (GPCR) signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the reassociation of G protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway.
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Introduction |
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Gradient sensing is mediated by G protein–coupled receptors (GPCRs) and associated signaling components that detect the spatiotemporal changes of chemoattractants and translate shallow gradients of chemoattractants into steep intracellular gradients of signaling components (Parent and Devreotes, 1999; Chung et al., 2001b; Funamoto et al., 2002; Iijima et al., 2002). Binding of cAMP to the GPCR cAR1 induces the dissociation of heterotrimeric G proteins into G
2 and Gß
subunits (Jin et al., 2000; Janetopoulos et al., 2001; Xu et al., 2005). Free Gß activates Ras, thereby leading to the activation of PI3K, which converts PI(4,5)P2 (PIP2) to PI(3,4,5)P3 (PIP3) in the plasma membrane (Li et al., 2000; Funamoto et al., 2001; Stephens et al., 2002; Sasaki et al., 2004; Wessels et al., 2004). The phosphatase PTEN acts as an antagonist of PI3K, dephosphorylating PIP3 to regenerate PIP2 (Funamoto et al., 2002; Iijima and Devreotes, 2002; Li et al., 2005). PIP3 mediates cellular processes by recruiting proteins with pleckstrin homology (PH) domains, such as cytosolic regulator of adenylyl cyclase (CRAC) and Akt/PKB, to the plasma membrane (Parent et al., 1998; Meili et al., 1999). Both CRAC and Akt/PKB play roles in the regulation of actin polymerization during chemotaxis (Meili et al., 1999; Comer et al., 2005). Recent progress in fluorescence microscopy has permitted measurements of the spatiotemporal changes of many signaling events in living cells with high spatiotemporal resolution required to test models of gradient sensing (Ueda et al., 2001; Sasaki et al., 2004; Xu et al., 2005).
There are several key features of gradient sensing. First, cells have the ability to spontaneously terminate responses under a sustained cAMP stimulation in a process called "adaptation" (Parent et al., 1998; Xu et al., 2005). Second, if cAMP is removed from adapted cells, the cells will enter a de-adaptation phase—a refractory period lasting several minutes during which the cells progressively regain their ability to respond to another cAMP stimulation (Dinauer et al., 1980a,b). Third, cells have the capability of translating shallow cAMP gradients across the cell diameter into highly polarized intracellular responses, a process called "amplification" (Parent and Devreotes, 1999; Servant et al., 2000; Chung et al., 2001a). To explain these features, it has been proposed that an increase in receptor occupancy activates two antagonistic signaling processes: a rapid "excitation" that triggers cell responses, such as the membrane accumulation of PIP3, and a slower "inhibition" that turns off those responses (Parent and Devreotes, 1999). Although many of the molecular mechanisms of the excitatory process have been identified, those of the inhibitory process have remained elusive.
The dynamic relationship between excitation and inhibition that leads to activation, adaptation, and amplification has been studied by direct visualization and quantitative analysis of the spatiotemporal changes in receptor occupancy, G protein dissociation, PI3K and PTEN distribution, and PIP3 level along the membrane (Xu et al., 2005; Meier-Schellersheim et al., 2006). Over the years, models have been proposed to explain how the excitatory and the inhibitory processes interact in cells responding to chemoattractants to achieve adaptation or amplification (Meinhardt, 1999; Parent and Devreotes, 1999; Postma and Van Haastert, 2001; Devreotes and Janetopoulos, 2003; Arrieumerlou and Meyer, 2005; Charest and Firtel, 2006; Levine et al., 2006; Meier-Schellersheim et al., 2006). Although inhibitors are essential components of all gradient sensing models, the spatial-temporal presence of inhibitors has not been examined experimentally.
In this study, we designed sequential stimulation protocols to detect temporal and spatial aspects of the inhibition process in single living cells. We found that repeated transient activations of cAR1 receptor trigger repetitive PHCrac-GFP membrane translocations without detectable refractory periods, demonstrating that a short pulse of cAR1 activation elevates excitation but little long-lasting inhibition. This result provides evidence that cAR1 activation induces an immediate excitation and a delayed recruitment of long-term inhibition leading to PIP3 accumulation. More significantly, we have revealed spatial distribution of the inhibition process induced by a cAMP gradient. Exposing a cell to a sustained cAMP gradient leads to a stable PHCrac-GFP accumulation in the front of the cell. We found that a sudden withdrawal of the cAMP gradient from this biochemically polarized cell leads to a rapid return of G protein activation, PTEN, and PIP3 distributions to basal levels around the cell membrane. However, there was a short time period during which reactivation of receptors and G proteins around the membrane induced a clear PIP3 response in the back but not the front of the cell. This inverted PIP3 response indicates that a cAMP gradient induces a stronger inhibition of PI3K in the front of a cell.
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24 s, a minimal time required for the cAMP concentration to return to its basal level between stimuli (Fig. 1 D). Sequential transient cAMP stimuli with as short as 24-s intervals generated repetitive transient responses of PHCrac-GFP translocation, and transient responses displayed kinetics without a refractory period (Fig. 1 D). Our data indicate that a transient receptor activation quickly activates excitatory pathways leading to an increase in PIP3, and upon cAMP removal, these pathways quickly return to prestimulated levels and can be activated again by another cAMP stimulation. Transient cAR1 activations do not signal long enough to substantially elevate the slower inhibition process from its basal level. Therefore, this result supports the idea that cAR1-mediated excitation and inhibition process increases and decreases by following a fast and a slow temporal mode, respectively.
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6 min (Devreotes and Steck, 1979; Devreotes, 1994), they were challenged with a uniformly applied cAMP stimulus. Interestingly, this induced an "inverted" response in which PHCrac-GFP transiently translocated to the back of the cells, demonstrating that the original front sides of the cells were less responsive to cAMP than were the back sides. Moreover, cells that had been exposed to gradients of various cAMP concentrations for the initial stimulus also exhibited inverted PHCrac-GFP responses upon a uniform cAMP stimulation (Fig. 2, B–G; and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200611096/DC1).
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2-CFP and YFP-Gß ("G cells") were suddenly exposed to 10 µM cAMP (Fig. 3, A and B), a saturating dose for cAR1, or 1 µM cAMP (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200611096/DC1). Addition of cAMP induced a rapid FRET loss, which reached a steady state in <20 s, indicating G protein dissociation (Fig. 3 B; Fig. S2) (Xu et al., 2005).
After the removal of cAMP, FRET returned to the prestimulus level in 60 s, indicating that the G proteins were completely reassociated. A second sudden exposure to the same concentration of cAMP triggered an instant FRET loss that displayed kinetics very similar to those in response to the first stimulation (Fig. 3 B; Fig. S2), demonstrating that cAR1 receptor and G proteins rapidly returned to their prestimulation states and could be fully reactivated within 60 s after the removal of cAMP.
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Spatiotemporal dynamics of PHCrac-GFP membrane translocation in PTEN null cells in response to cAMP stimuli
cAR1 activates an excitatory signaling branch that induces PTEN to translocate from the membrane to the cytosol and also elevates an inhibitory mechanism that allows cytosolic PTEN to return to the membrane (Funamoto et al., 2002; Iijima and Devreotes, 2002). To determine spatiotemporal changes in PIP3 in the cells lacking PTEN, we measured kinetics of PHCrac-GFP membrane translocation in pten– cells and compared the kinetics to those in wild-type (WT) cells (Xu et al., 2005; Meier-Schellersheim et al. 2006), in response to uniformly applied cAMP and a cAMP gradient (Fig. 4).
WT and pten– cells expressing PHCrac-GFP were stimulated uniformly with cAMP (1 µM) at 0 s (Fig. 4 A). The cAMP-triggered PHCrac-GFP membrane translocation is fast and transient in WT cells. In contrast, the response in pten– cells was clearly slower, peaking in 12 s and returning to prestimulus levels in more than 40 s (Fig. 4 A). When the cells were suddenly exposed to a gradient (Fig. 4 B), membrane translocations of PHCrac-GFP occurred initially in both front and back regions in both WT and pten– cells. However, the kinetics of PHCrac-GFP translocation in the front was clearly abnormal in pten– cells. There was no clear decrease in PHCrac-GFP amount at the front for more than 150 s, which differs from the biphasic response in WT cells (Fig. 4 B). We also examined kinetics of the PIP3 in response to the removal of cAMP stimuli in pten– cells (Fig. 4 C). After cells were exposed to a gradient, PHCrac-GFP accumulates in the front regions of WT or pten– cells. Upon a removal of the gradient at 0 s, PHCrac-GFP gradually returned to the cytosol. The returning process was clearly slower in pten– cells than in the WT cells, whose t1/2 were 22 s and 14 s, respectively (Fig. 4 C).
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80 s without over-accumulating in the front (Fig. 5).
Furthermore, reapplying a uniform stimulus (Fig. 5, A–D) or gradient (Fig. 5, E–H) of cAMP induces PTEN translocation with kinetics (Fig. 5) similar to those observed in the cells that had not previously been stimulated (Meier-Schellersheim et al., 2006), indicating that the cAR1-controlled regulatory components of PTEN returned to their "resting" states and PTEN molecules in both the front and back were responsive to a second cAMP stimulation when the inverted PHCrac-GPF response occurred. Therefore, the excitatory and the inhibitory mechanisms that control PTEN membrane distribution are not the likely explanation for this inverted response.
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1 or G9 subunits are not essential for cAR1-mediated PHCrac-GFP responses9 and G1-mediated PLC pathways in D. discoideum have been shown to function as negative regulators in the cAR1-mediated signaling (Bominaar and Van Haastert, 1993; Brzostowski et al., 2004). To test whether either pathway is essential for the gradient sensing, we examined PHCrac-GFP responses in G9 and G1 null cells (Fig. 6). We measured PHCrac-GFP membrane translocation by monitoring intensity changes of GFP fluorescence in the cell membrane (Xu et al., 2005). In response to a uniform stimulation, the spatiotemporal kinetics of PHCrac-GFP membrane translocation in either g9– or g1– were similar to those in the WT cells (Fig. 6, A and C).
When the g9– or g1– cells were suddenly exposed to stable cAMP gradients, PHCrac-GFP translocation, as in WT cells, consisted of two phases, an initial transient translocation around the cell membrane followed by a second phase producing a highly polarized distribution (Fig. 6, B and D). Because our observed dynamics in both mutant cells are similar to those displayed in WT cells (Xu et al., 2005; Meier-Schellersheim et al. 2006), we suggest that G1 or G9 controlled signaling are not essential inhibitory mechanisms for cAR1-mediated gradient sensing.
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activates Ras that stimulates a small amount of preexisting, membrane-associated PI3K. The resulting actin polymerization leads to recruitment of additional PI3K from cytosol to the membrane, thereby increasing the amount of active PI3K (Sasaki et al., 2004). In Latrunculin-treated cells, PI3Ks were uniformly distributed around the membrane of the cells even when they were exposed to the cAMP gradient (Sasaki et al., 2004; unpublished data). Therefore, under our experimental condition, we monitored the spatiotemporal regulations of PI3K activity without complications from the second layer of actin-dependent PI3K recruitment. In addition to the signaling pathway leading to PI3K activation, the cAMP receptor also regulates another pathway mediating the redistribution of membrane-bound PTEN, which is important for the proper directional response of PIP3. In pten– cells, PHCrac-GFP was still able to accumulate in the front when the cells were exposed to a cAMP gradient (Janetopoulos et al., 2004; Sasaki et al., 2004). We found that the crescents of PHCrac-GFP in pten– cells were broader than those formed in WT cells (Fig. 7, E and F; at time 0), as previously described (Janetopoulos et al., 2004; Sasaki et al., 2004). Furthermore, the directions of the crescents, unlike those in WT cells, did not always perfectly point to the direction of the gradient (Fig. 7 F; at time 0). These results indicate, as expected, that a cAMP gradient-induced PTEN redistribution ensures the PIP3 response in the restricted front region, and this directional response was not precise without PTEN. However, after a withdrawal of the cAMP gradient, PHCrac-GFP returned to cytosol. More importantly, the second cAMP gradient induced the inverted PHCrac-GFP membrane translocation in pten– cells (Fig. 7, E and F), indicating that a cAMP gradient-induced asymmetrical inhibition occurred in the absent of PTEN. Collectively, our results suggest that the previous gradient induced an asymmetrically distributed and locally controlled inhibition and this localized inhibition acts on the signaling pathway between free Gß to PI3K.
Kinetics of the asymmetrical inhibition induced by a cAMP gradient
We examined the temporal appearance and disappearance of the gradient-induced asymmetrical inhibition (Fig. 8; Fig. S4, B and C, available at http://www.jcb.org/cgi/content/full/jcb.200611096/DC1). We found that a brief gradient stimulation of 50 s was not sufficient to induce an inverted PHCrac-GFP response (Fig. 8, A and B; Fig. S4 B).
Thus, exposure to a stable gradient for 2 min is needed to establish an asymmetrical inhibition. Furthermore, cells that were removed from a gradient for 6 min and rechallenged with either uniform cAMP stimulation or a cAMP gradient displayed a noninverted PHCrac-GFP translocation response as in naive cells (Fig. 8, C and D; Fig. S4 C), indicating that asymmetrical inhibition disappears within 6 min after the gradient is removed.
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We have constructed a quantitative model for cAR1-mediated signaling network (Meier-Schellersheim et al., 2006). The model, which includes receptor-mediated and locally controlled inhibitory mechanisms that regulate PI3K and PTEN (Fig. 9 B), simulates experimentally determined dynamics of receptor activation, G protein dissociation, PTEN membrane localization, and PIP3 accumulation (Meier-Schellersheim et al., 2006).
For example, in response to a uniform cAMP stimulus, the model generates a transient PIP3 response that quickly returns to the resting stage (adaptation). When exposed to a cAMP gradient, a cell generates a steeper PIP3 gradient by initially inducing a PIP3 increase followed by a PIP3 decrease around the membrane, and then producing a highly polarized distribution of PIP3 in 120 s (amplification) (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200611096/DC1). During the amplification process, the membrane-bound PTEN gradually translocates from the front to the back, while the amount of the membrane-associated PI3K remains the same around the membrane (Fig. 9 C). Temporal changes in PI3K activity in the front and back, which cannot be directly visualized, have been simulated by the model based on dynamics of PIP3 and membrane-bound PTEN (Meier-Schellersheim et al., 2006; Fig. S5). Previous study and our measurements indicated that when a cell reaches the "polarized" steady state, the amount of PI3K in the front is almost equal to that in the back (Sasaki et al., 2004). PIP3 is at a higher steady-state level in the front. However, this level does not continue to increase (
PIP3/t = 0) in spite of a lower level of PTEN. At the same time, in the back, a higher level of PTEN does not result in a continuous decrease in the PIP3 level (PIP3/t = 0). Two possible models may explain different steady states of PIP3 in a polarized cell. First, PI3K activity is stronger than that of PTEN in the front, and PIP3s are continually produced. The PIP3 level remains steady in the front because it diffuses fast enough to be degraded by PTEN that is enriched in the back, which is expected from models containing only globe inhibition mechanisms (Parent and Devreotes, 1999; Iglesias and Levchenko, 2002; Iijima et al., 2002). Second, balances between the activities of PI3K and PTEN have been reached in both the front and back, and the balances are achieved by a stronger inhibition of PI3K activity in the front, which have been proposed in our model that includes local inhibition mechanisms (Xu et al., 2005; Meier-Schellersheim et al., 2006). Because different proposed mechanisms could lead to high chemotactic sensitivity in theory (Meinhardt, 1999; Postma and Van Haastert, 2001; Devreotes and Janetopoulos, 2003; Arrieumerlou and Meyer, 2005; Levine et al., 2006; Meier-Schellersheim et al., 2006), we designed experiments to determine which inhibitory mechanisms are likely used in GPCR-mediated chemosensing. In this study, we revealed spatiotemporal features of an inhibitory process that acts locally on the activation pathway between Gß and PI3K.
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to PI3K (Fig. 9, B and C). The relatively slower recovery of the responsiveness in PIP3 production in the front of the cell revealed that the inhibitory effect diminished slowly. The fact that the PHCrac-GFP inversion response was also observed in pten– cells indicated that the recruitment of inhibitors does not depend on PTEN (Fig. 7, E and F). Postma et al. (2004) reported that cells that were stimulated with a sustained uniform cAMP field did not result in a clear decrease in PIP3 production to another cAMP stimulation, suggesting that the recovery period is very short in a cell that has adapted to a uniform cAMP concentration. It is possible that a high level of the inhibitor is only induced by a cAMP gradient at the front of a cell where the PIP3 level is high but not by a uniform cAMP around the cell membrane where the PIP3 level remains low. We can only speculate on this point before the putative inhibitors in GPCR-mediated chemosensing network are identified.
The inhibition has been assumed to be "global" or uniformly distributed throughout the plasma membrane even when a cell is exposed to a cAMP gradient (Parent and Devreotes, 1999; Iglesias and Levchenko, 2002; Iijima et al., 2002; Devreotes and Janetopoulos, 2003; Janetopoulos et al., 2004). Our findings demonstrate that the concept of a purely global inhibition cannot be reconciled with the observed spatial distributions of some inhibitory mechanisms. The inverted PIP3 response upon restimulation indicates that a sustained cAMP gradient induces an asymmetrically distributed inhibition that acts on the signaling pathway between G protein and PI3K (Fig. 7). This inhibition is stronger in the front of the cell. The spatiotemporal features of the inhibition can shed light on unknown molecular mechanisms. Based on the fast-diffusive-inhibition models, small molecules, such as Ca2+ or cGMP, were suggested to be candidate inhibitors, which have not been verified by experiments. The "local excitation and global inhibition" model assumes the presence of a negative regulator, and suggests that it is likely to be PTEN. Based on our detailed spatiotemporal dynamics of PTEN and PIP3, our computational model showed that PTEN alone cannot fully explain the experimentally determined dynamics (Meier-Schellersheim et al., 2006). We proposed, in addition to PTEN, other inhibitory mechanisms that may involve reversible modifications of components in the pathway from free Gß to Ras and then to PI3K. Previous studies in mammalian GPCR signaling indicated several inhibitory components. After GPCR activation, free Gß dimers interact with the receptor-associated kinase GRK2, blocking Gß signaling (Lodowski et al., 2003). GPCR activation can also induce a translocation of a RasGAP, which binds to PIP3, to inner membrane deactivating Ras thereby inhibiting PI3K (Lockyer et al., 1999). In D. discoideum, it has been shown that a sustained cAR1 activation, which triggers a persistent G protein dissociation, induces a transient activation of RasG, which activates PI3K (Sasaki et al., 2004). The transient nature of RasG activation is consistent with the idea that the cAR1 activation also recruits inhibitors to the membrane to shut down signals from free Gß to Ras activation. Our computational model is able to simulate the observed spatiotemporal dynamics of known components in adaptation and in gradient sensing by including these putative inhibitor(s) (Meier-Schellersheim et al., 2006). Therefore, we propose that the inhibition process is performed by these negative regulators acting locally on the PI3K signaling branch and those on PTEN branch, which act in concert to control the spatiotemporal dynamic of PIP3 around the cell membrane. Future studies are needed to identify inhibitors involved in the GPCR-mediated chemosensing network.
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Materials and methods |
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2CFP and YFPGß (Xu et al., 2005); and pten–, g1– and g9– cells expressing PHCrac-GFP were developed to the chemotactic stage. Cells were plated on a 1-well chamber for the microinjector delivered cAMP stimulation (Nalge Nunc International), allowed to adhere to the cover glass for 10 min, and then covered with additional DB buffer. Live cells were imaged using a Zeiss Laser Scanning Microscope, LSM 510 META, with a 40x NA 1.3 DIC Plan-Neofluar objective. To monitor cAMP and PHCrac-GFP, PTEN-GFP, PI3K1-GFP cells were excited with two laser lines, 488 nm for GFP and 543 nm for Alexa 594, a water-soluble fluorescence dye. Images were simultaneously recorded in three channels. Channel one: fluorescent emissions from 505–530 nm for GFP (green); channel two: emissions from 580–650 nm for Alexa 594 (red).
Generation and measurement of applied cAMP stimulations
The temporal-spatial intensity changes of Alexa 594 and cells expressing PHCrac-GFP, PTEN-GFP, or PI3K1-GFP were directly imaged using a confocal microscope with Z-axis resolution of 2 µm. Fluorescence intensities of Alexa 594 and GFP within the focal plane were simultaneously recorded in two different channels. To establish a steady gradient, we set an external supply pressure to 70 hPa (Femtojet and micromanipulator 5171; Eppendorf) to ensure the injection of a constant and small volume of cAMP and Alxea 594 into a one well chamber. Under this condition, a stable gradient was established within 100 µm around the tip of the micropipette. To suddenly expose a cell to a stable gradient, a micropipette filled with a mixture of cAMP and 0.1 g/µl Alexa 594 linked to a FemtoJet was positioned 1,000 µm away from the cells, and then was quickly moved to a position within 100 µm to the cells. During the experiments, we only changed the distance between the micropipette and the cells. The speed of the movement determines how fast a stable gradient can form around a cell (Xu et al., 2005). To withdrawal a gradient, the micropipette was quickly moved away from a cell.
FRET measurement
Using a spectral confocal fluorescence microscope (LSM510 META), we measured intensity decrease of acceptor (YFP) and increase of donor (CFP) in response to stimuli. We monitored intensity changes of CFP (donor) and (YFP) acceptor following a stimulation using a time-lapse acquisition of Lambda Stacks. The cells were excited with a 454-nm laser line and the spectral emissions in each pixel of the fluorescence images were simultaneously recorded in 8 channels, each with a 10-nm width, from 464 to 544 nm. To separate multi-fluorescence signals, each of the fluorescence images was collected using Lambda Stack acquisition. The spectral emissions of fluorescence images were simultaneously recorded in a CHS-1 from 464 nm to 544 nm. The spectra of the cells expressing CFP, YFP or GFP only were obtained and used as the references for the Linear Unmixing Function. The digitally separated images of CFP and YFP of the G cells, and GFP of the PH cells were obtained. The intensities of each fluorophore in the regions of interest in the time-lapse experiments were measured, normalized, and expressed as a function of time in responses to cAMP stimulations, using the software of LSM510 META (Xu et al., 2005).
Imaging and data processing
Images were processed and analyzed by the LSM 510 META software, and converted to TIFF files by the Adobe Photoshop software. All frames of any given series were processed identically. Selected frames of the series were assembled as montages using Photoshop 7.0. Quantification of fluorescence intensities of Alexa 594, GFP, CFP, and YFP in the regions of interest was performed using the LSM 510 META software.
Online supplemental material
Fig. S1 shows inverted PIP3 responses. Fig. S2 shows FRET measurement of G protein dissociation and association and redissociation. Fig. S3 shows reapplied a cAMP gradient induced an inverted PHCrac-GFP membrane translocation. Fig. S4 A shows G9 null cells detect cAMP gradient normally. Fig. S4 B and C show kinetics of the formation the asymmetrically distributed inhibition. Fig. S5 shows PI3K activity, membrane-bound PTEN and the resulting dynamics of PIP3 in a cell when it is exposed to a cAMP gradient in a computer simulation and a schematic representation of the signaling network that describes spatiotemporal changes. Videos 1 and 2 show uniformly applied cAMP stimulation triggered inverted PHCrac-GFP translocation. Video 3 shows simultaneously measurement of G protein activation in the front and back of a cell and the inverted PHCrac-GFP response. Video 4 shows dynamics of PTEN in a cell upon a withdrawal of a cAMP gradient and then reapplied the gradient. Videos 5 and 6 show a cAMP gradient induced the inverted PHCrac-GFP membrane translocation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200611096/DC1.
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Acknowledgments |
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9– cells expressing PHCrac-GFP. We thank D. Hereld, A. Kimmel, J. Brozostowski, S. Ou, and X. Xiang for critical comments; and S. Pierce and R. Germain for support. This study is supported by NIAID/NIH intramural funds.
Submitted: 16 November 2006
Accepted: 6 June 2007
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References |
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Arrieumerlou, C., and T. Meyer. 2005. A local coupling model and compass parameter for eukaryotic chemotaxis. Dev. Cell. 8:215–227.[CrossRef][Medline]
Bominaar, A.A., and P.J. Van Haastert. 1993. Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C. J. Cell Sci. 104:181–185.[Abstract]
Brzostowski, J.A., C.A. Parent, and A.R. Kimmel. 2004. A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement. Genes Dev. 18:805–815.
Charest, P.G., and R.A. Firtel. 2006. Feedback signaling controls leading-edge formation during chemotaxis. Curr. Opin. Genet. Dev. 16:339–347.[CrossRef][Medline]
Chung, C.Y., S. Funamoto, and R.A. Firtel. 2001a. Signaling pathways controlling cell polarity and chemotaxis. Trends Biochem. Sci. 26:557–566.[CrossRef][Medline]
Chung, C.Y., G. Potikyan, and R.A. Firtel. 2001b. Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol. Cell. 7:937–947.[CrossRef][Medline]
Comer, F.I., and C.A. Parent. 2002. PI 3-kinases and PTEN: how opposites chemoattract. Cell. 109:541–544.[CrossRef][Medline]
Comer, F.I., C.K. Lippincott, J.J. Masbad, and C.A. Parent. 2005. The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis. Curr. Biol. 15:134–139.[CrossRef][Medline]
Condeelis, J., R.H. Singer, and J.E. Segall. 2005. The great escape: when cancer cells hijack the genes for chemotaxis and motility. Annu. Rev. Cell Dev. Biol. 21:695–718.[CrossRef][Medline]
Devreotes, P.N. 1994. G protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron. 12:235–241.[CrossRef][Medline]
Devreotes, P., and C. Janetopoulos. 2003. Eukaryotic chemotaxis: distinctions between directional sensing and polarization. J. Biol. Chem. 278:20445–20448.
Devreotes, P.N., and T.L. Steck. 1979. Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response. J. Cell Biol. 80:300–309.
Devreotes, P.N., and S.H. Zigmond. 1988. Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. Annu. Rev. Cell Biol. 4:649–686.[CrossRef][Medline]
Dinauer, M.C., T.L. Steck, and P.N. Devreotes. 1980a. Cyclic 3',5'-AMP relay in Dictyostelium discoideum IV. Recovery of the cAMP signaling response after adaptation to cAMP. J. Cell Biol. 86:545–553.
Dinauer, M.C., T.L. Steck, and P.N. Devreotes. 1980b. Cyclic 3',5'-AMP relay in Dictyostelium discoideum V. Adaptation of the cAMP signaling response during cAMP stimulation. J. Cell Biol. 86:554–561.
Funamoto, S., K. Milan, R. Meili, and R.A. Firtel. 2001. Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in Dictyostelium. J. Cell Biol. 153:795–810.
Funamoto, S., R. Meili, S. Lee, L. Parry, and R.A. Firtel. 2002. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell. 109:611–623.[CrossRef][Medline]
Gerisch, G. 1982. Chemotaxis in Dictyostelium. Annu. Rev. Physiol. 44:535–552.[CrossRef][Medline]
Iglesias, P.A., and A. Levchenko. 2002. Modeling the cell's guidance system. Sci. STKE. 2002:RE12.[Medline]
Iijima, M., and P. Devreotes. 2002. Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell. 109:599–610.[CrossRef][Medline]
Iijima, M., Y.E. Huang, and P. Devreotes. 2002. Temporal and spatial regulation of chemotaxis. Dev. Cell. 3:469–478.[CrossRef][Medline]
Janetopoulos, C., T. Jin, and P. Devreotes. 2001. Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science. 291:2408–2411.
Janetopoulos, C., L. Ma, P.N. Devreotes, and P.A. Iglesias. 2004. Chemoattractant-induced phosphatidylinositol 3,4,5-trisphosphate accumulation is spatially amplified and adapts, independent of the actin cytoskeleton. Proc. Natl. Acad. Sci. USA. 101:8951–8956.
Jin, T., N. Zhang, Y. Long, C.A. Parent, and P.N. Devreotes. 2000. Localization of the G protein betagamma complex in living cells during chemotaxis. Science. 287:1034–1036.
Levchenko, A., and P.A. Iglesias. 2002. Models of eukaryotic gradient sensing: application to chemotaxis of amoebae and neutrophils. Biophys. J. 82:50–63.[Medline]
Levine, H., D.A. Kessler, and W.J. Rappel. 2006. Directional sensing in eukaryotic chemotaxis: a balanced inactivation model. Proc. Natl. Acad. Sci. USA. 103:9761–9766.
Li, Z., H. Jiang, W. Xie, Z. Zhang, A.V. Smrcka, and D. Wu. 2000. Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. Science. 287:1046–1049.
Li, Z., X. Dong, Z. Wang, W. Liu, N. Deng, Y. Ding, L. Tang, T. Hla, R. Zeng, L. Li, and D. Wu. 2005. Regulation of PTEN by Rho small GTPases. Nat. Cell Biol. 7:399–404.[CrossRef][Medline]
Lockyer, P.J., S. Wennstrom, S. Kupzig, K. Venkateswarlu, J. Downward, and P.J. Cullen. 1999. Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo. Curr. Biol. 9:265–268.[CrossRef][Medline]
Lodowski, D.T., J.A. Pitcher, W.D. Capel, R.J. Lefkowitz, and J.J. Tesmer. 2003. Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma. Science. 300:1256–1262.
Meier-Schellersheim, M., X. Xu, B. Angermann, E.J. Kunkel, T. Jin, and R.N. Germain. 2006. Key role of local regulation in chemosensing revealed by a new molecular interaction-based modeling method. PLoS Comput. Biol. 2:e82.[CrossRef][Medline]
Meili, R., C. Ellsworth, S. Lee, T.B. Reddy, H. Ma, and R.A. Firtel. 1999. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 18:2092–2105.[CrossRef][Medline]
Meinhardt, H. 1999. Orientation of chemotactic cells and growth cones: models and mechanisms. J. Cell Sci. 112:2867–2874.[Abstract]
Murphy, P.M. 1994. The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12:593–633.[CrossRef][Medline]
Parent, C.A., and P.N. Devreotes. 1999. A cell's sense of direction. Science. 284:765–770.
Parent, C.A., B.J. Blacklock, W.M. Froehlich, D.B. Murphy, and P.N. Devreotes. 1998. G protein signaling events are activated at the leading edge of chemotactic cells. Cell. 95:81–91.[CrossRef][Medline]
Pollard, T.D., and G.G. Borisy. 2003. Cellular motility driven by assembly and disassembly of actin filaments. Cell. 112:453–465.[CrossRef][Medline]
Postma, M., and P.J. Van Haastert. 2001. A diffusion-translocation model for gradient sensing by chemotactic cells. Biophys. J. 81:1314–1323.[Medline]
Postma, M., J. Roelofs, J. Goedhart, H.M. Loovers, A.J. Visser, and P.J. Van Haastert. 2004. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches. J. Cell Sci. 117:2925–2935.
Sasaki, A.T., C. Chun, K. Takeda, and R.A. Firtel. 2004. Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J. Cell Biol. 167:505–518.
Servant, G., O.D. Weiner, P. Herzmark, T. Balla, J.W. Sedat, and H.R. Bourne. 2000. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 287:1037–1040.
Stephens, L., C. Ellson, and P. Hawkins. 2002. Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr. Opin. Cell Biol. 14:203–213.[CrossRef][Medline]
Ueda, M., Y. Sako, T. Tanaka, P. Devreotes, and T. Yanagida. 2001. Single-molecule analysis of chemotactic signaling in Dictyostelium cells. Science. 294:864–867.
Van Haastert, P.J., and P.N. Devreotes. 2004. Chemotaxis: signalling the way forward. Nat. Rev. Mol. Cell Biol. 5:626–634.[CrossRef][Medline]
Wessels, D., R. Brincks, S. Kuhl, V. Stepanovic, K.J. Daniels, G. Weeks, C.J. Lim, G. Spiegelman, D. Fuller, N. Iranfar, et al. 2004. RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum. Eukaryot. Cell. 3:646–662.
Xu, J., F. Wang, A. Van Keymeulen, P. Herzmark, A. Straight, K. Kelly, Y. Takuwa, N. Sugimoto, T. Mitchison, and H.R. Bourne. 2003. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell. 114:201–214.[CrossRef][Medline]
Xu, X., M. Meier-Schellersheim, X. Jiao, L.E. Nelson, and T. Jin. 2005. Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium. Mol. Biol. Cell. 16:676–688.
Zigmond, S.H. 1978. Chemotaxis by polymorphonuclear leukocytes. J. Cell Biol. 77:269–287.
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