Exocyst is involved in polarized cell migration and cerebral cortical development Kresimir Letinica,b, Rafael Sebastianc, Derek Toomreb, and Pasko Rakica,1 aDepartment of Neurobiology and Kavli Institute for Neuroscience and bDepartment of Cell Biology, 333 Cedar Street, Yale University School of Medicine, New Haven, CT 06510; and cCenter for Computational Imaging and Simulation Technologies in Biomedicine, Universitat Pompeu Fabra, Carrer Roc Boronat, 138-08018 Barcelona, Spain
Contributed by Pasko Rakic, May 7, 2009 (sent for review January 14, 2009)
Neuronal migration is essential for proper development of the cerebral cortex. As a first step, a postmitotic cell extends its leading process, presumably by adding new membrane at the growing tip, which would enable directed locomotion. The goal of the present study was to determine if biosynthetic exocytic pathway is polarized in migrating cells and whether polarized exocytosis promotes directed cell migration. A promising candidate for controlling the spatial sites of vesicle tethering and fusion at the plasma membrane is a protein complex called the exocyst. We found that cell migration in a wound assay, as well as cortical neuronal migration during embryonic development was impaired when the exocyst was disturbed. By combining TIRF microscopy and a stochastic model of exocytosis, we found that vesicle exocytosis is preferentially distributed close to the leading edge of polarized cells, that the exocytic process is organized into hotspots, and that the polarized delivery of vesicles and their clustering in hotspots depend on the intact exocyst complex. The exocyst complex seems to achieve this spatial regulation by determining the sites at the membrane where secretory vesicles tether. Thus, our study supports the notion that polarized membrane traffic regulated by the exocyst is an essential component of cell migration and that its deficit may lead to cortical abnormalities involving cortical neuronal malpositioning. astrocyte 兩 cell polarization 兩 membrane trafficking 兩 neuronal ectopia 兩 neuronal migration
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lthough most cells in the animal’s body are mobile, the migration has a particular significance in the laminated structures of the mammalian brain, where the neuronal position determines its function (1). Apart from the need to recognize pathways, a migrating cell has to translocate its nucleus and the surrounding cytoplasm within the growing leading process (2). Thus, one of the prerequisites for neuronal migration is the extension of the leading process in one specific direction. Remarkably, very simple but clever experiments in the 1970’s indicated that migrating cells grow predominantly by adding new membrane at the tip of the extending process (3). Electron microscopic reconstructions of migrating cortical neurons revealed membrane surface expansion at the leading edge in the course of their migration and thus, further supported the notion of a selective addition of new membrane at the tip (4). Cortical cells originate during development from progenitors located close to the brain ventricles, in the so-called ventricular zone (1, 5). From there, they need to migrate increasingly long distances to the superficial layers of the cortex where they form the cortical plate, or the prospective cortex. This process needs to be precisely coordinated, considering that newly born neurons have to bypass the older ones and settle in the uppermost cortical layers, a process called inside-out migration (6, 7). Disturbance of neuronal migration may results in cortical malformations, or fine abnormalities leading to neurological and psychiatric syndromes (5, 8, 9). Early electron microscopic studies have shown that, not only the microtubule cytoskeleton polarizes in migrating neurons (10), but also their membrane surface area increases 11342–11347 兩 PNAS 兩 July 7, 2009 兩 vol. 106 兩 no. 27
as they start to migrate from the ventricular zone (4). Studies have well established that long-distance migration of neurons, which consists of leading process extension and nuclear translocation, depends on the polarization of the centrosome and microtubules (10–13). Numerous studies implicated a variety of adhesion and cytoskeletal proteins and related signaling pathways in the process of neuronal migration (10–17). However, the role and cellular and molecular mechanisms of polarized membrane traffic remain mostly unknown. Considering the importance of neuronal migration for the normal cortical development as well as for the pathogenesis of neurological and psychiatric disorders (5, 8, 9), we decided to examine whether a disturbance of membrane traffic affects the positioning of neurons in the cortical layers. Exocytosis is fundamental for the proper function of all cells, tissues, and organs. It falls into 2 categories: regulated and constitutive secretion (18). Regulated secretion is essential for neurotransmission and release of exocrine and endocrine factors. Constitutive secretion, by contrast, occurs in most cells and is not under tight temporal control. Polarized cells, such as epithelial cells and neurons, use spatial control of where vesicles fuse to produce plasma membranes of different composition and function (19). A long postulated hypothesis is that vesicle delivery to the leading edge can promote cell migration (20). The exocytosis near the front edge is conceptually attractive as it could promote focal membrane expansion (19–22). Increasing evidence suggests that membrane traffic and its spatial reorganization are indeed involved in the cellular processes of cytokinesis (23) and cell migration (24–27). Direct observations of exocytic events in migrating fibroblasts support this notion (28). Research in this area is of great interest as altered cell migration is associated with metastasis and invasion of cancer cells. Studies have only recently examined the molecular mechanisms of membrane traffic in migrating non-neuronal cells. Expression of a kinase-dead mutant of protein kinase D1, which perturbs post-Golgi membrane trafficking, inhibited fibroblast migration (24) as did perturbation of SNARES (26). Knockdown of RalB and exocyst proteins impaired migration of NRK cells (27), but the mechanisms remain unknown. Recently it became evident that a protein complex called the exocyst is an excellent candidate for determining the spatial location of vesicle fusion at the plasma membrane (29). Studies in yeast and mammalian cells have provided considerable evidence that the exocyst is essential for polarized membrane traffic (29–37). The exocyst presumably functions before the SNAREs but after vesicles exit the Golgi and are trafficked to the cell periphery (30–32). A recent study showed that RalB regulates exocyst function and is involved in directional movement (27). Despite evidence for a role of the exocyst in the exocytic process, it remains unknown where and when precisely this complex acts, Author contributions: K.L., D.T., and P.R. designed research; K.L. and D.T. performed research; K.L. and R.S. analyzed data; and K.L. and P.R. wrote the paper. The authors declare no conflict of interest. 1To
whom correspondence should be addressed. E-mail:
[email protected].
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Fig. 1. Wound assay. Representative images of control (Upper Left) and Sec8-depleted (Upper Right) cells migrating into the wound; the wound is delimited by the square (dashed line). (Lower Right) The graph shows mean (⫾ SD) wound width (pixels) 12-h after wounding. (Lower Left) A western blot showing an effect of Sec8 RNAi on the level of endogenous Sec8 protein in NRK cells; tubulin level serves as a control. **, P ⬍ 0.01.
largely due to a lack of good assays to monitor exocytosis at the single vesicle level. In the present study, we examined how a disturbance of the exocyst affects spatial sites of exocytosis in polarized cells and the rate of cell migration in vitro and in vivo, using a variety of approaches. We used total internal ref lection f luorescent (TIRF) microscopy (38–42) in combination with RNAi against Sec8, the most frequently studied exocyst subunit. As a next step, we capitalized on the existence of a dominant negative mutant of the Exo70 exocyst subunit, which allowed us to test the importance of the exocyst directly in migrating cortical neurons during embryonic development. Results and Discussion As a first step, to determine if the exocyst is necessary for cell migration in general, we depleted Sec8, an exocyst component, from normal rat kidney cells (NRK) and examined their migration in a wound essay. NRK cells were a useful model system in this type of experiment because they migrate at a fast rate in a wound assay. A significant decrease in cell migration into the wound was found upon Sec8 depletion (Fig. 1), which is consistent with a recent report (27). This finding suggested that exocyst might be important for maintaining a proper cell migration rate. To examine the role of the exocyst in the developing mouse central nervous system, we tried but could not successfully to introduce Sec8 RNAi into migrating neurons by microinjecting and electroporating the embryonic forebrain. Instead, we took Letinic et al.
Fig. 2. Representative images of cortical sections, illustrating the distribution of migrating cells across the cortical layers (VZ, ventricular zone; IZ, intermediate zone; CPL, lower half of the cortical plate; CPU, upper half of the cortical plate). (Upper) The schematic drawing illustrates neurons (green) migrating along long radial glial fibers (blue) from the VZ to the CP. (Lower) The graph shows proportions of cells in different cortical layers of the control brains (red) and brains expressing ExoDN (green).
advantage of the existence of a dominant negative mutant of Exo70 (ExoDN), a major exocyst component which directly binds Sec8 and targets the whole complex to the plasma membrane. We performed in utero microinjection of ExoDN (GFP as a marker) into the mouse cerebral ventricles in embryonic day 15 mice, followed by electroporation (transfection) of cells, which are close to the ventricular surface. We allowed mouse embryos to develop inside the womb for 4 days, after which we killed them. We then fixed and examined their brains for the location of cells positive for GFP. We noticed a significant difference in the distribution of migrating neurons between the brains electroporated with a control plasmid and the brains electroporated with Exo70DN (a likelihood ratio based test, G-test; the value of test statistics G ⫽ 2737, P ⫽ 0.000) (Fig. 2). In the control brains, most cells (number of cells counted ⫽ 10.448) were located in the upper cortical plate. In the brains transfected with ExoDN, most cells (number of cells counted ⫽ 15.172) were still in the intermediate zone (Fig. 2). Furthermore, the odds that a cell assumed a location in the ventricular and intermediate zones, as opposed to the cortical plate, increased by a factor of 3.31 in brains transfected with ExoDN (P ⫽ 0.000). PNAS 兩 July 7, 2009 兩 vol. 106 兩 no. 27 兩 11343
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Fig. 3. TIRFM imaging of astrocytes. (A) A TIRF image of a polarized astrocyte, whose cell body is outlined in (B), where the arrow shows the direction of migration, and the orange solid circle marks the location of the cell nucleus (N). (A) Several VCVG-labeled vesicles are circled in red. (C) Selected frames of a VSVG-labeled vesicle undergoing fusion. As a vesicle enters the evanescent field and tethers to the membrane, it exponentially increases in brightness (see the 3D-intensity profile). As it fuses, the brightness of the spot increases, but later, as the dye continues to spread, and the diameter of the bright area increases, its intensity diminishes. This ‘‘flash’’ is a characteristic signature of fusions. (D) A western blot showing an effect of Sec8 RNAi on the level of endogenous Sec8 protein in astrocytes; tubulin level serves as a control.
These finding strongly indicated that the exocyst complex is necessary for the proper rate of neuronal migration during cortical development. The major limitation of the use of TIRFM is the object (eg, cell, vesicle) size. Not only are the vesicles in migrating neurons tiny, but the cell surface and volume are extremely small as well. For this reason, we could observe neither the labeled vesicles nor the leading process of migrating neurons using TIRF microscopy. Numerous studies have demonstrated that fundamental cellular and molecular mechanisms implicated in the control of cell migration are shared between neurons and astrocytes (11– 13). One example is the molecular control of microtubule polarization during cell migration. Moreover, cortical astrocytes share common origin with cortical neurons and migrate along similar routes in vivo. They have a trailing and leading edge, which are, in vitro, much larger than those of migrating neurons. We thus believe that migrating neurons and astrocytes also share similar membrane traffic regulatory mechanisms. In contrast to NRK cells, astrocytes are very flat and large and have easily observable secretory vesicles in TIRFM. While NRK are fast moving and ideal for a migration assay, astrocytes are slower but ideal for imaging vesicle exocytosis. To investigate whether the exocyst controls the sites of vesicle fusion, we mapped exocytosis in polarized astrocytes treated with control or Sec8 RNAi. We infected astrocytes with an adenovirus expressing vesicular stomatitis virus glycoprotein (VSVG). We imaged both polarized and nonpolarized cells using TIRF microscopy (Fig. 3). Cells at the edge of the wound are highly polarized; they have an elongated, often triangular cell body with a wide leading edge and a narrow trailing edge. The nucleus is located in the back of the cell within the trailing edge; the Golgi apparatus positions itself between the nucleus and the leading edge. We imaged nonpolarized cells in confluent cultures. Polarized cells depleted of Sec8 were, in terms of their shape, cell area and distribution of major cytoskeletal elements (actin and microtubules) indistinguishable from the control cells. We cannot exclude the possibility that microtubule dynamics, ie, fine movements of 11344 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0904244106
microtubules right beneath the cell membrane, might be changed in exocyst-depleted cells. Using custom-made programs, fusion event were marked, to obtain spatial-temporal maps of fusions, and sorted into 8 equally spaced bins along the main axis of the cell (bin 8 is closest to the leading edge; the ‘rear’ in the case of CNP cells was arbitrary). We visually observed a striking difference in the distribution of fusion events between polarized and nonpolarized control cells, as well as between control and sec8-depleted polarized cells (Fig. 4A). As expected, in control nonpolarized cells (CNP), fusions (n ⫽ 292) were relatively uniformly distributed, with no obvious hot spots (Fig. 4B). In control polarized cells (CP), fusions (n ⫽ 554) were preferentially localized in the front of the cell, with highest numbers in bins 6 and 7, followed by bin 8 (Fig. 4B). In Sec8-depleted cells (S8P), fusions (n ⫽ 390) were much more uniformly distributed, with the highest proportion located in bins 4/5 (Fig. 4B). A statistical G-test demonstrated a highly significant difference between CP and CNP cells (test statistics, G ⫽ 174.23, P ⫽ 0.000), as well as between CP and S8P cells (G ⫽ 172.78, P ⫽ 0.000). Sec8-depletion significantly increased the odds that an event occurs in the rear half of the cell by a factor (called the odds ratio) of 10.5 (P ⫽ 0.000). No difference in the odds was found between S8P and CNP cells (odds ratio was 0.98, P ⫽ 0.878). These results strongly suggest that the exocyst controls polarized delivery of vesicles during cell migration. Sec8-depletion could affect the sites of exocytosis by disturbing the tethering process or by disturbing the fusion process that follows tethering. We compared the distribution of tethering sites in S8P and CP cells (n ⫽ 1170 for S8P; n ⫽ 760 for CP; Fig. 4C) and found a striking difference (G ⫽ 181.1, P ⫽ 0.000). There was a striking resemblance in the distribution of tethering and fusion sites within each group (Fig. 4C), but not between the groups. These findings strongly indicate that the distribution of tethering sites dictates the distribution of fusion sites and that the effect of Sec8-depletion on the fusion sites can be at least in part explained by its effect on the tethering sites. Letinic et al.
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Since fusion events often clustered in small domains of the membrane, which we call hotspots, we tested whether Sec8depletion causes a randomization of the locations of fusion events. We modeled the exocytic process as a spatial point process, ie, a random variable producing points at random locations representing the location of events (42). A homogenous Poisson process is a completely random point process, in which the number of events in nonoverlapping spatial regions is independent and the probability of an event is the same at all locations. In contrast, in a clustered process, the location of points depends on the location of nearby points. We used L-function, a variant of Ripley’s K-function, to compare the observed exocytic point process with a set of spatial Poisson point processes generated by Monte Carlo simulations. This test gives an idea of how far the actual distribution of fusion events is from Poisson distribution. We observed that the exocytic Letinic et al.
process is strongly clustered in CP cells, but either weakly clustered, or not significantly different from completely random in S8P cells (Fig. 5). The difference between these 2 groups of cells was highly significant (bootstrap analysis, P ⫽ 0.02). The exocytic process in CNP cells was not significantly different from the one in S8P cells (P ⫽ 0.96), and was weakly significantly different from the one in CP cells (P ⫽ 0.06) (Fig. 5). These findings indicate that the exocyst mediates spatial clustering of fusion events into hotspots. A week clustering exists in some nonpolarized cells, but clustering becomes much more prominent in polarized cells. By using TIRF microscopy and spatial statistical tools, we found strong evidence for the notion that vesicle exocytosis is polarized toward the leading edge in polarized/migrating cells. A previous study (28) has found that in polarized fibroblasts more fusion events occur in the front two-thirds of the cell, which PNAS 兩 July 7, 2009 兩 vol. 106 兩 no. 27 兩 11345
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Fig. 4. (A) Spatial maps and histograms of fusion events in representative control polarized (Middle), nonpolarized (Top), and Sec8-depleted polarized (Bottom) cells. Front and rear of each cell is indicated, as well as the direction of migration (1). (B) Line plots of the distribution of fusion events across the cell surface that was divided into bins. (C) Line plots of the distribution of both fusion and tethering events in control and Sec8-depleted polarized cells.
Fig. 5. Representative L-curves of control polarized (A), control nonpolarized (B), and Sec8-depleted polarized (C) cells. The gray-lined area between the envelopes (dotted gray lines) corresponds to a set of simulated Poisson/random point processes and is labeled ‘‘randomness’’. The colored curves correspond to observed data points (each curve represents 1 cell). L-curves of the CP cells are shifted upwards relative to those of the other 2 groups. Different groups are compared by using bootstrap analysis, shown in (D). Bootstrap regions (95% confidence intervals) are significantly different between the CP and the other 2 groups, whereas they largely overlap for CNP and S8P.
together with our finding strongly supports that exocytosis is polarized in cell migration. A finding of the present study is that the exocytic process is organized into dynamic clusters or hotspots and that both the polarized delivery of vesicles and their clustering depend on the exocyst complex, which controls the spatial exocytic pattern in part by determining the sites at the plasma membrane to which vesicles tether. We propose that by regulating the spatial sites of vesicle tethering and fusion near the leading edge, the exocyst is involved in the fine regulation of the rate of cell migration. The finding that depleting exocyst proteins results in a diminished migration supports this notion. While the role of integrin, actin, and Rho-family GTPases in cell migration is well-established, results provided here indicate that spatial control of vesicle traffic plays a key regulatory role in cell migration. Polarized exocytosis and dynamic clusters may provide means to deliver new membrane, cytoskeletal, and cell adhesion elements critical for the cell to extend the leading process and to respond to environmental cues. In conclusion, insight into the regulation of membrane trafficking during neuronal migration will be essential for understanding of both normal cerebral cortical development and the pathogenesis of congenital cortical abnormalities that lead to neurological and psychiatric syndromes. For example, our finding that interference with Exo70 results in visible neuronal ectopias opens up a possibility that exposure to various drugs that interfere with exocytosis during pregnancy may cause an overt or subtle, but functionally significant neuronal malpositioning. Materials and Methods Migration Assays and RNAi. We used normal rat kidney (NRK) cells and astrocytes, which represent commonly used migration assays. Briefly, around 70% confluent monolayers of NRK cells were treated for 3 days with control or Sec8 siRNA, wounded and 12 h later wound closure was quantitated (phase contrast images of a dozen random fields were acquired and used to obtain the mean gap). Astrocytes were treated with siRNA in the same way, infected with temperature-sensitive YFP-tagged vesicular stomatitis virus glycoprotein 11346 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0904244106
(tsVSVG-YFP) adenovirus (40 – 42), blocked at 39 °C overnight, scratch wounded, and 4 h later released at 32 °C and imaged by TIRFM. Quantification showed ⬇70 –90% knockdown of endogenous protein for both cell types. TIRF microscopy, mapping of fusion sites, and spatial statistical analysis were described in previous work (42). We microinjected a GFP-plasmid (control) alone, or in combination with a plasmid expressing Exo70 DN, into the embryonic day 15 mouse brains. This procedure was followed by electroporation, after which the embryos were allowed to develop inside the uterus and killed 4 days later. Brains were examined for the presence of GFP-fluorescence, cut on a cryostat, and mounted on microscope slides. Visually we observed a consistent difference between the control and treated group; more than 3 brains per group (control- and Exo70DN-transfected) were randomly picked from a larger number of brains for detailed analysis. Cell counting was done using Neurolucida program. TIRFM. We imaged astrocytes by TIRFM (objective-type, 60⫻, 1.45 NA, excitation ⫽ 488 nm) at 37 °C at 2 Hz. Cells were imaged after release from a temperature block for 1–1.5 h. TIRFM data were uploaded into Matlab software (matlab 6.5; The MathWorks) for the detection of exocytic vesicles. All vesicle information (time, position) was stored and used to generate temporal and spatial maps of vesicle docking and fusion. For every cell, we observed its exocytic pattern. Statistical Analysis. Proportions of cells in different embryonic cortical zones was calculated by using the total number of labeled cells as the denominator. Statistical G-test was done in Excel. G-test is based on log-likelihood ratio and is defined as G ⫽ ⌺iOi䡠In(Oi/Ei), where Oi is the frequency observed and Ei is the frequency expected in any given bin. Under the null hypothesis that the observed frequencies come from a distribution with the designated expected frequencies, the distribution of G is approximately 2. Logistic regression to estimate the odds ratios was done in Stata 10 (StataCorp). Stochastic Model. We considered the spatial locations of fusion events and analyzed exocytosis as a spatial point process. As a functional descriptor of spatial arrangement, we used the so-called K-function. K-function values are calculated by putting circles of increasing radius size on each individual fusion and counting the number of neighboring fusions that fall inside the circle. For aggregated patterns, each event is likely to be surrounded by many other events, so for a circle of small radius s, K(s) will be relatively large. If fusions are randomly distributed, or if they are regularly spaced, each event is likely to be
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surrounded by empty space. In this case, for a circle of small radius s, K(s) will be relatively small. Our goal was to evaluate whether the locations of fusion events were randomly distributed, ie, whether they could be considered a realization of Poisson point process. In the Poisson process, the points in space do not have any relationship to each other and environment. Monte Carlo method was used to achieve this goal. Random Poisson processes were generated in the statistical program R using Monte Carlo simulation. A variancestabilizing version of Ripley-K function, called L-function, conveys the same information but illustrates more clearly the differences between any 2 spatial patterns. L-functions were calculated both for the simulated Poisson patterns and for the observed exocytic patterns. Using the simulated processes, we obtained an upper and a lower range of L-values (envelopes marked by gray dashed lines in the graphs in Fig. 5 A–C). When the L-curve of an observed data point (1 cell) falls within the envelopes of randomness, this fact suggested a
random distribution of fusions. In contrast, if it falls outside and above the envelopes, this fact suggested a clustered process. Bootstrap method was used to estimate standard errors and confidence intervals and for a comparison of different groups of cells.
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ACKNOWLEDGMENTS. We thank T. Koleske, P. Novick, and A. Barron for helpful discussions; S. C. Hsu (University of Medicine and Denistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ) for providing antibodies against exocyst proteins; A. R. Saltiel (University of Michigan, Ann Arbor, MI) for providing Exo70DN; and R. H. Scheller (Stanford University School of Medicine, Stanford, CA) for providing Sec8-GFP. K.L. is a predoctoral fellow of the Howard Hughes Medical Institute. This work was supported by funding from the National Institutes of Health (NS038296; NS14841, to P.R.).
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