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Architecture of cannabinoid signaling in mouse retina

Cones, Bosko, Hashish Kos

Try out PMC Labs and tell us what you think. Learn More. Louis, Missouri, USA. Understanding the physiology of complex relationships between components of signaling pathways and the actin cytoskeleton is an important challenge. CD2AP is a membrane scaffold protein implicated in a variety of physiological and disease processes. The physiological function of CD2AP is unclear, but its biochemical interactions suggest that it has a role in dynamic actin assembly. Here, we report that CD2AP functions to facilitate the recruitment of actin capping protein CP to the Src kinase substrate, cortactin, at the cell periphery, and that this is necessary for formation of the short branched filaments that characterize lamellipodium formation and are required for cell migration. Superresolution fluorescence microscopy demonstrated that the efficient colocalization of CP and cortactin at the cell periphery required CD2AP. As both cortactin and CP function to enhance branched actin filament formation, CD2AP functions synergistically to enhance the function of both proteins. Our data demonstrate how the interplay between specialized actin regulatory molecules shapes the actin cytoskeleton. Animal cells control their shape and move about by regulating the assembly of actin filaments in space and time. Unique cell shapes and movements are defining features of differentiated cell function, and they change in the course of pathological processes. While a number of molecular components capable of interacting with actin and controlling its polymerization have been identified, the field has a very limited understanding of how this complex array of components, with a multitude of biochemical interactions, works together to assemble actin and produce the forces that dictate shape and power movement. Using biochemical approaches, we identified several actin regulatory proteins as interacting with the membrane scaffold CD2AP 1. However, the physiological significance of these interactions is not known. Here, we address this question with experiments that reveal that interactions of CD2AP with CP and cortactin indeed have physiological significance, and we elucidate how these interactions lead to spatial and temporal recruitment and assembly of the actin filament network that drives cell shape changes at the periphery of motile cells. CD2AP is a signaling scaffold protein originally discovered to be necessary for the formation of the actin-based immunological synapse 2. CD2AP is expressed in a wide variety of cells, with higher levels in epithelial cells, immune cells, and neurons 3. Loss of CD2AP from glomerular epithelial cells, also known as podocytes, leads to renal failure 4. CD2AP was also found to associate with the actin regulatory protein cortactin in biochemical studies 9. The physiological significance of this interaction, however, remains unclear. Loss of CD2AP led to loss of CP recruitment to the cell periphery, and this depended on the direct interaction between the two proteins. Together, our results suggest that the formation of lamellipodia is mediated by a complex of cortactin, CD2AP, and CP. The formation of this complex may help to create a feedback loop that promotes the formation of a branching network of actin filaments in lamellipodia. Green fluorescent protein GFP fused to actin was subcloned into a retroviral expression vector driven by the pMX promoter Louis, MO Anticortactin MAb 4F11 was purchased from Millipore. The virus secreted by these cells was used to infect the podocytes. Podocytes were grown in mm tissue culture dishes to confluence, and a scratch wound was created with a pipette tip. For video microscopy, a Cascade charge-coupled device CCD camera was used. Video was compiled using Metamorph software Universal Imaging Corp. The number of cells that migrated toward the wound was counted for each cell type. Cells were allowed to attach for 5 min and spread for 5 or 15 min. They were then fixed and imaged by phase-contrast microscopy. Frames were collected every 3 s for 7 min. Cells were grown on acid-washed, 2. The images were acquired at ms intervals. The intensity of GFP-actin before bleaching was set at 1, and the bleaching was set at 0. The ratio of the recovered to the original intensity was plotted against time in seconds using Microsoft Excel. The intensity of the bleached area was corrected for photobleaching. Using confocal microscopy, lamellipodia were identified as thin protrusive sheets of membrane at the periphery of the cell. As it is sometimes difficult to distinguish between membrane ruffles and lamellipodia, only cells exhibiting GFP signals in clearly discernible lamellipodia were counted as positive. For each group, averages of 80 to cells were analyzed and quantified. The stochastic optical reconstruction microscopy STORM setup and imaging were similar to those described before Antibody control was prepared by adhering a diluted mixture of anti-mouse Cy3-Aconjugated and anti-rabbit Aconjugated secondary antibodies to a coverglass flow cell. All imagings were performed by inverting coverslips onto a glass slide with imaging buffer ratio of PBS, 1 M mercaptoethylamine \\\\\\\\\\\\\[pH 8. Excess buffer was drained and the edges sealed with nail polish before image acquisition. STORM images of entire cells were reconstructed using Gaussian fitting to find the center position of single molecules. The cross-correlation function represents the probability of finding a second channel localization at a certain distance, r , from the first channel. A value of 1 represents a random distribution between the two channels, and values higher than 1 suggest a correlation of two channel molecules at a certain distance. Colocalization analysis was performed using Manders' overlap coefficient, which is based on Pearson's coefficient Briefly, the Manders' overlap coefficient represents the degree of colocalization between the two channels. Since CP is important in the formation of actin-based structures at the cell periphery 21 , 22 , our initial experiments examined whether CD2AP plays a role in CP recruitment to the periphery of spreading podocyte cells. Wild-type podocytes were allowed to adhere to glass and spread for up to 30 min. The transfected cells were identified using immunofluorescence. Thus, efficient recruitment of CP to the cell periphery at early time points during cell spreading might be mediated by CD2AP. Thus, CD2AP facilitates the recruitment of CP to the periphery of spreading cells, apparently through a direct molecular interaction. Since CP is important in the formation of actin-based structures during cell spreading, we assessed the morphology of CD2AP-deficient cells during cell spreading by phase-contrast microscopy Fig. As illustrated in Fig. Thus, cell spreading is affected by the loss of CD2AP, with changes in the morphology of the actin-based structures at the spreading edge of the cells. Interestingly, the observed increase in ruffling and filopodial patterns is similar to the effects described for the loss of CP 21 , CD2AP is required for efficient cell spreading and migration. A Wild-type and CD2AP-deficient podocytes were plated onto glass coverslips and fixed after 5 or 15 min. Cells were classified into three different morphologies, examples of which are shown in Fig. The smooth-edge phenotype is characterized by a flat, evenly spreading leading edge. The ruffling phenotype is characterized by phase-dense undulations of the peripheral membrane. The filopodial phenotype is characterized by finger-like protrusions at the periphery of the cell. B Quantification of the smooth-edge, ruffling, and filopodial phenotypes of wild-type and CD2AP-deficient podocytes. Phenotypes were assessed by phase-contrast microscopy of fixed podocytes at 5 and 15 min after making contact with a glass slide. Images were scored blinded and classified as one of the three phenotypes. Most of the wild-type podocytes displayed smooth-edge and ruffling phenotypes. In contrast, the CD2AP-deficient podocytes had a higher propensity to spread with ruffling phenotype. C Wound healing assay. A wound was inflicted with a p pipette tip. At The white lines represent the boundaries of the original wound. D Column bar graph showing the number of cells that migrated toward the wound. A box was drawn at the boundaries of the original wound, and cells within the box after Fewer CD2AP-deficient podocytes than wild-type cells had migrated into the wound. While wild-type cells exhibited a smooth and circumferential distribution of actin during spreading, the CD2AP-deficient cells exhibited a patchy and irregular pattern of actin see Movies S1 and S2 in the supplemental material. These results confirm that CD2AP is important for the structure and dynamics of the actin cytoskeleton at the edge of the cell during spreading. Alterations in the actin-based protrusive structures at the cell edge might be expected to have effects on cell migration. We assessed the role of CD2AP in cell migration using a standard scratch-wound healing assay. Wild-type or CD2AP-deficient podocytes were plated on tissue culture dishes data not shown or on glass coverslips Fig. A scratch wound was created on the monolayer using a pipette tip, and the cells were monitored for 16 to 24 h by video microscopy. While wild-type cells closed the wound in 14 to 18 h, loss of CD2AP led to slower migration of cells into the wound based on measurements of the thickness of the wound Fig. Video microscopy also demonstrated that while the wild-type podocytes moved in a coordinated sheet-like manner, the CD2AP-deficient podocytes moved more discordantly see Movies S3 and S4 in the supplemental material. We confirmed that the phenotype was specific to CD2AP and not a clone-specific difference by using two different lentivirus-expressed shRNAs to downregulate CD2AP expression in wild-type cells data not shown. We bleached an area at the periphery of the cell and monitored the fluorescence recovery over the following 60 s Fig. We also bleached an area in the center of the cell near the nucleus. Here, we saw no difference between wild-type and CD2AP-deficient cells in terms of mobile fraction or recovery time Fig. Thus, CD2AP promotes the dynamic turnover of actin filaments at the periphery of the cell, consistent with a role in actin assembly and actin-based motility. CD2AP affects actin dynamics at the cell periphery. The half-life of actin recovery in wild-type podocytes is To investigate the mechanism of how CD2AP is recruited to the periphery of the cell, we analyzed the localization of various deletion mutants of CD2AP. CD2AP contains several potential protein interaction domains, including three Src homology 3 SH3 domains at the amino terminus, a proline-rich region, and a coiled-coil domain at the carboxy-terminal half. Both were fused to GFP. Thus, the C-terminal half of CD2AP is necessary and sufficient for its localization to the cell periphery. Twenty-four hours after transfection, cells were replated on glass-bottom dishes and images were acquired using confocal microscopy. Pro, proline-rich sequence; CC, coiled-coil sequence. The asterisk indicates the binding site for CP. It was previously reported that cortactin can interact with the C-terminal half of CD2AP 9 , but the function of this interaction was not determined. Cortactin is also important for the assembly of actin-based structures that contribute to cell migration We therefore considered whether cortactin mediates the recruitment of CD2AP to the periphery. We first examined the localization of cortactin in wild-type and CD2AP-deficient podocytes. Interaction of CD2AP with cortactin is required for lamellipodial localization. Twenty-four hours after transfection, cells were replated and images were acquired using confocal microscopy. In the overlay image, CD2AP is green and cortactin is red. No significant membrane localization was seen. Thus, CD2AP recruitment to the cell periphery requires the SH3 domain of cortactin as well as the second proline-rich motif of CD2AP, most likely via a direct interaction of those regions of the proteins. A Efficient knockdown of cortactin expression using two different shRNA constructs. The percentage of cells with the smooth-edge phenotype is shown. These results demonstrate that interactions with both cortactin and CP are necessary for CD2AP function at the cell periphery during spreading. We therefore used superresolution fluorescence imaging to determine whether we could detect these complexes at the cell periphery. Superresolution imaging allows for subdiffraction-limit imaging of cellular macromolecular complexes 30 — We hypothesized that if CD2AP functions to link cortactin with CP, the colocalization between the molecules would be higher in the wild type Fig. B and D A region of the lamellipodia, boxed in panels A and C, is shown at a resolution high enough to allow the position of individual proteins to be determined and the correlation analysis to be performed. Bar, nm. Colocalization analysis of CP and cortactin at the cell periphery in wild-type versus CD2AP knockout cells left bars compared to a random position in the cytoplasm right bars was first normalized to the density of cortactin and then analyzed using the Manders' overlap coefficient. Analysis was performed on 6 randomly selected cells and 10 regions in each cell. Error bars represent standard deviations. F Cross-correlation analysis of CP and cortactin in wild-type versus CD2AP cells was performed on areas of the cell periphery compared to the cytoplasm, with two different secondary antibodies as a negative control. Each point represents the probability that a spot in the second channel is present within a distance of r of the spot in the first channel. Values of 1 y axis are indicative of a random distribution, while values greater than 1 indicate colocalization. Data were acquired and analyzed as described in Materials and Methods. Both analyses showed that at the cell periphery there was significantly greater colocalization of GFP-CP with cortactin in wild-type Fig. In addition, coclustering of CP with cortactin was much lower in the center of the cell. Interestingly, the density of cortactin recruitment was higher in wild-type cells than in CD2AP-deficient cells Fig. Since CP is involved in actin assembly at the plasma membrane 34 , 35 , we asked here whether CD2AP affected actin assembly and dynamics at the plasma membrane. Supporting an important role for CD2AP in CP function, we found that the recruitment of CP to the cell periphery was defective in CD2AP-deficient cells, and this correlated with defects in cell spreading and cell motility. The switch between filopodia and lamellipodia has been proposed to depend on the local activity of CP Because of the ability of CP to stop growth at the fast-growing barbed end of actin filaments, CP was originally considered a negative regulator of actin polymerization 35 , Indeed, acute inhibition of CP in a small region of the cell leads to a local increase in actin polymerization. However, greater complexity was revealed in other studies. One potential molecular explanation for this result is that the level of CP controls the length of actin filament branches in the network and that networks require optimal branch lengths in order to support the force produced by growing actin filament ends as they push outward on the plasma membrane. We previously showed that CD2AP can inhibit the actin capping activity of CP, but the effect is partial and not complete 1. Consistent with this, structural studies demonstrate that the CP-binding motif found in CD2AP binds at a site distinct from the actin-binding surface 1 , 8 , suggesting that CP bound to CD2AP still retains actin capping activity. For these reasons, we speculate that the CP that is targeted to the cell periphery by CD2AP is not inhibited and caps barbed ends. CD2AP has a particularly critical role in the podocyte, as revealed by the significant kidney phenotype that develops in the CD2AP-knockout mouse 4. Podocytes, however, express CD2AP but not CIN85 41 , so the absence of both proteins may be required to produce the phenotypes seen here. Indeed, the migration and the spreading defects that we observed were not apparent in CD2AP-deficient mouse embryonic fibroblasts that express CIN85 data not shown. Because the ability of podocytes to spread and cover exposed areas of the glomerular basement membrane may be an important response to kidney injury, the defect in CP recruitment and lamellipodia formation described here may explain the kidney failure that develops in the CD2AP-deficient mice 4. Our finding that cortactin can recruit CD2AP, which then recruits CP, provides novel insight into the mechanism of actin polymerization at the cell periphery. Cortactin was first discovered as a major substrate for the Src tyrosine kinase By coupling modifiers of actin polymerization via its SH3 domain, cortactin may function to generate different types of actin structures in the cell. Our data showing that cortactin then recruits CD2AP and CP demonstrate how it uses its SH3 domain of cortactin to specifically enhance and sustain branched actin fiber assembly in lamellipodia. By recruiting CP, CD2AP may generate a positive-feedback loop, with increased branched actin filaments allowing for the recruitment of additional cortactin molecules. This could explain the decreased density of peripheral cortactin in CD2AP-deficient cells data not shown. Protein-protein interactions are easily detected using biochemical approaches, but verifying their existence in cells remains a significant challenge. While two-color immunofluorescence colocalization studies are often employed to support a specific interaction, these studies are limited by the low resolution of light microscopic imaging and often the very broad cellular expression patterns of a specific protein. Electron microscopy with immunogold labeling can overcome some of the resolution issues, but this method is highly cumbersome and the number of proteins labeled is often highly limited. One utility of superresolution methods like STORM and PALM 32 , 47 is that they can robustly localize thousands of single molecules at nanometer resolution and allow for rigorous statistical analytic tools to be applied to the problem of colocalization Using two analytic approaches, a colocalization and a cross-correlation analysis, we could easily show that the colocalization of CP and cortactin at the cell periphery required the presence of CD2AP, a conclusion that was not apparent by visual inspection of the images. In summary, we have demonstrated that two proteins, CP and cortactin, previously known to be important for actin assembly and cell migration, form a complex with CD2AP at the periphery of the cell. Branched actin filaments are also important in vesicular trafficking and endocytosis, so these findings provide insight into other possible roles for CD2AP in the function of normal and diseased cells and tissues. Published ahead of print 22 October National Center for Biotechnology Information , U. Journal List Mol Cell Biol v. Mol Cell Biol. Cooper , b and Andrey S. Shaw a, c. John A. Andrey S. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address correspondence to Andrey S. Shaw, ude. All Rights Reserved. This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplemental material. Abstract Understanding the physiology of complex relationships between components of signaling pathways and the actin cytoskeleton is an important challenge. Cell culture and transfection. Cell motility, cell spreading, and video microscopy. TIRF microscopy. 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