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. 2013 Dec 9;203(5):815-33.
doi: 10.1083/jcb.201304143.

FSGS3/CD2AP Is a Barbed-End Capping Protein That Stabilizes Actin and Strengthens Adherens Junctions

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Free PMC article

FSGS3/CD2AP Is a Barbed-End Capping Protein That Stabilizes Actin and Strengthens Adherens Junctions

Vivian W Tang et al. J Cell Biol. .
Free PMC article

Abstract

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell-cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell-cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity.

Figures

Figure 1.
Figure 1.
Identification of two actin-regulating activities at the adherens junctional complex. (a–c) Flow chart represents the overall scheme for biochemical analysis of actin-regulating factors on junction-enriched membranes. (a) Identification of a detergent extractable inhibitory factor on native membranes. Addition of Alexa Fluor 647–labeled monomeric actin to native membranes results in actin assembly associated with the junctional complexes (white arrowheads). Treatment of native membranes with zwitterionic detergent CHAPS or nonionic detergent TX-100 yielded brighter actin puncta (orange and red arrowheads, respectively). (b) Identification of a high-salt/detergent extractable activating factor on native membranes. Treatment of native membranes with high salt followed by CHAPS diminished actin incorporation at membrane puncta (blue arrowheads). Rescaling of the original image shows the presence of very dim fluorescent actin puncta (yellow arrowheads). (c) Identification of a high-salt extractable inhibitory factor on native membranes. Treatment of native membranes with high salt increased actin incorporation at membrane puncta (green arrowheads) when compared with native membranes (purple arrowheads) supplemented with α-actinin-4. (d) Quantitation of actin incorporation at junctional puncta in untreated native membranes and membranes treated with detergents CHAP (P < 0.0001) or TX-100 (P < 0.0001). (e) Quantitation of actin incorporation at junctional puncta in untreated native membranes and membranes treated with high salt (P < 0.0001). (f) Quantitation of actin incorporation at junctional puncta in high salt–treated or sequentially high salt– and CHAPS-treated membranes supplemented with α-actinin-4 (P < 0.0001).
Figure 2.
Figure 2.
Purification and identification of junction-associated actin-regulatory factor complexes using a target bait-based, cross-linking approach. (a) Negative staining of MDCK membranes showing 6 nm immunogold labeling for α-actinin-4 (blue arrowheads) at sites of actin incorporation (red circle) and filament association (yellow arrowheads). (b) Immunofluorescence staining of TX-100 extracted MDCK cells for β-catenin, α-actinin-4, and actin (phalloidin) showing colocalization at junctional puncta (white arrowheads). A subset of β-catenin staining has no α-actinin-4 or actin (yellow arrowheads). (c) Western blots of MDCK cell extracts for E-cadherin and α-actinin-4 showing resistance to detergent extraction by TX-100. (d) Negative staining of TX-100–treated membranes showing actin filament (red arrowhead) associated with macromolecular complexes (blue arrowhead). (e) Negative staining of CHAPS-treated membranes showing actin filament (red arrowhead) associated with macromolecular complexes (blue arrowhead). (f) Negative staining of TX-100–resistant junctional complex (yellow outlines) showing 6 nm immunogold labeling for α-actinin-4 (yellow arrowheads). (g) Addition of cross-linker–derivatized recombinant α-actinin-4 to high salt stripped membranes results in targeting of α-actinin-4 to a membrane junctional complex that supports actin assembly. Activation of a cross-linker functional group by UV results in covalent attachment of α-actinin-4 to proteins in close proximity. Non-cross-linked proteins are removed by detergent solubilization. Identification of CD2AP, EVL, radixin, and vinculin by mass spectroscopy of gel bands from cleaved α-actinin-4 cross-linked complexes is shown. Coomassie blue staining of SDS-PAGE gel shows purification steps (lanes 1–4).
Figure 3.
Figure 3.
Identification of CD2AP and EVL as the actin-regulating activities at the adherens junctional complex. (a) Biochemical reconstitution of actin assembly on high salt/CHAPS-treated membranes with α-actinin-4 and EVL. (b) Quantitation of actin puncta showing recovery of actin assembly on high salt/CHAPS-treated membranes (LPHSCh) supplemented with α-actinin-4 and EVL (P < 0.0001) but not with α-actinin-4 or EVL alone. (c) Biochemical reconstitution of actin assembly on high salt–treated membranes with α-actinin-4 alone or together with CD2AP. (d) Quantitation of actin puncta showing inhibition of actin assembly by CD2AP (P < 0.0001). (e) F-actin spin-down assay showing cosedimentation of α-actinin-4 and CD2AP with F-actin. (f) CD2AP did not interfere with α-actinin-4 recruitment to the membrane junctional complex. High salt–stripped membranes were incubated with rhodamine-labeled α-actinin-4 in the absence or presence of 1 µM CD2AP. (g) Actin depolymerization assay by dilution of membrane junctional actin puncta into buffer in the presence or absence of the severing protein cofilin.
Figure 4.
Figure 4.
CD2AP inhibits actin polymerization and depolymerization by capping actin barbed ends. (a) Pyrene-actin spectroscopy assay showing a dose-dependent decrease in spontaneous actin polymerization rate by CD2AP. Pyrene fluorescence was measured at 30-s intervals to obtain the trace. A representative trace from four experiments is shown. (b) Two-color actin single filament imaging showing addition of rhodamine-labeled (red) monomeric actin to preformed rhodamine green-labeled (green) actin filaments. (c) Two-color actin single filament imaging showing inhibition of red actin addition by preincubating green actin filaments with CD2AP. (d) Two-color actin single filament imaging showing addition of red actin after preincubating green actin filaments with the C-terminal half of CD2AP. (e) Two-color actin filament imaging of limulus parallel actin bundles showing inhibition of red actin addition to the fast-growing barbed ends by preincubating green actin filaments with CD2AP. (f) Pyrene-actin spectroscopy assay in the presence of monomer sequestration protein, vitamin D–binding protein (VDBP), showing suppression of actin depolymerization by CD2AP. Pyrene fluorescence was measured at 30-s intervals to obtain the trace. A representative trace from two experiments is shown. (g) Single filament imaging showing actin depolymerization upon dilution into buffer. (h) Single filament imaging showing inhibition of actin depolymerization by preincubating filaments with CD2AP.
Figure 5.
Figure 5.
CD2AP is targeted to the adherens junction along with E-cadherin after a calcium-switch assay. Incubation of MDCK cells without calcium for 3 h results in diffuse cytoplasmic localization of CD2AP that is distinct from E-cadherin localization. Upon replenishment of calcium, CD2AP and E-cadherin appeared at nascent cell–cell contacts within 30 min. By 1 h, most cells reformed cell–cell contacts with E-cadherin and CD2AP localization at the junction.
Figure 6.
Figure 6.
CD2AP localizes to discreet junctional puncta in polarized MDCK cells. (a) Single deconvolved optical section showing colocalization of CD2AP with E-cadherin and actin (phalloidin). (b) Single deconvolved optical z sections scanning from the apical (0 µm) to lateral (0.8 µm) junction. Yellow arrowheads point to colocalization of CD2AP puncta with E-cadherin and actin. Purple arrowheads point to colocalization of CD2AP puncta with E-cadherin in the absence of actin. White arrowheads point to puncta with E-cadherin alone. (c) Single deconvolved optical section showing colocalization of CD2AP with α-actinin-4, E-cadherin, and actin (yellow arrowheads). A subset of E-cadherin puncta has α-actinin-4 and actin without CD2AP (white arrowheads), whereas another subset of E-cadherin puncta has CD2AP and actin without α-actinin-4 (purple arrowheads). There is also a population of CD2AP not associating with α-actinin-4, E-cadherin, or actin (orange arrowhead). (d) Single deconvolved optical section showing colocalization of CD2AP with E-cadherin at latrunculin-resistant actin clusters (yellow arrowheads).
Figure 7.
Figure 7.
CD2AP is required for actin stability at the adherens junction of polarized MDCK epithelial cells. (a) Projection of 40 deconvolved optical z slices spanning the apical 8 µm of cells showing decreased actin accumulation (yellow arrowheads) at cell–cell contacts in CD2AP knockdown cells (white asterisks) stained for E-cadherin, CD2AP, actin (phalloidin), and DNA (DAPI). (b) Western blots of total cell extracts showing normal levels of E-cadherin, α-actinin-4, and actin in stable CD2AP knockdown cell lines, ShCD2AP1 and ShCD2AP2. Molecular weight markers are 150 kD for CD2AP, α-actinin-4, and E-cadherin blots and 50 kD for the actin blot. (c) Single deconvolved optical z slice at the apical region of cells showing knockdown of CD2AP (white arrowheads and outlines) within a monolayer of cells expressing normal levels of CD2AP (yellow arrowheads and outlines). (d) Quantitation of junctional staining of E-cadherin and CD2AP showing a correlation between CD2AP levels and E-cadherin levels in parental but not CD2AP knockdown cells. R is the correlation coefficient. Representative data from four separate experiments are shown. (e) Quantitation of junctional staining of CD2AP and actin (phalloidin) showing a correlation between CD2AP levels and actin levels in both parental and CD2AP knockdown MDCK cells. Representative data from four separate experiments (n = 4) are shown. (f) Quantitation of junctional staining of E-cadherin and actin (phalloidin) showing a lack of correlation between E-cadherin levels and actin levels in both parental and CD2AP knockdown MDCK cells. Representative data from four separate experiments (n = 4) are shown. (g) Projection of 10 deconvolved optical z slices spanning the apical 2 µm of cells showing decreased actin levels at latrunculin-resistant puncta in CD2AP knockdown cells. (h) Quantitation of actin levels at latrunculin-resistant puncta in parental and CD2AP knockdown cells (P < 0.0001). Representative data from four separate experiments (n = 4) are shown.
Figure 8.
Figure 8.
CD2AP is required for the epithelial monolayer to withstand mechanical stress. (a) Projection of 70 deconvolved optical z slices of the entire 14-µm height of polarized MDCK cells showing a missing cell (white asterisks) within a cell monolayer transfected with CD2AP shRNA. (b) Phase-contrast microscopy showing a cell in mitosis (chromosome condensation, blue arrowheads) detaching from neighboring cells (red arrowheads) within a monolayer of cells transfected with CD2AP shRNA. (c) Time-lapse images of phase-contrast microscopy showing cells tearing away (red arrowheads) from each other within a monolayer of cells transfected with CD2AP shRNA. (d) BSA flux assays showing similar monolayer permeability between parental, CD2AP knockdown, and α-actinin-4 knockdown cells. (e) Occludin localization at the tight junction is normal in CD2AP knockdown cells. (f) Logistics of applying mechanical stress at intercellular junctions for the analysis of epithelial cohesion. (g) A pressure chamber apparatus that can generate hydraulic pressure across a cell monolayer to induce stress at intercellular junctions. (h) Pressure chamber apparatus is attached to a syringe for pumping fluid and a pressure gauge to monitor applied pressure. Transwell supports were held in position during pressure application by lids and screws at the top. (i) Schematics for the operation of the pressure chamber apparatus. Buffer is pumped through channels connecting the basal chambers to produce hydrostatic pressure. (j) Permeability assays showing increased tracer flux with increasing applied hydrostatic pressure to MDCK monolayers. (k) Permeability assays showing a low tolerance level and decreased resilience to mechanical stress in CD2AP knockdown cells. (l) Permeability assays showing greater increases in tracer permeability in CD2AP knockdown cells than in parental cells after mechanical stress. (m) E-cadherin localization at the cell–cell junction of MDCK cells showing intact monolayer after mechanical stress. (n) E-cadherin localization at the cell–cell junction of CD2AP knockdown cells showing holes (asterisks) in the monolayer after mechanical stress. All error bars indicate standard errors.
Figure 9.
Figure 9.
CD2AP suppresses cell motility and wound-induced cell migration. (a) Time-lapse phase-contrast microscopy showing migration of CD2AP knockdown cells into free space, whereas parental MDCK cells are relatively stationary. (b) Time-lapse phase-contrast microscopy showing wound-induced migration of 2 d postconfluent MDCK cell monolayer. (c) Quantitation of migration speed in MDCK cells showing decreased migration rate as a function of time (P < 0.0001). Representative data from three separate experiments (n = 3) are shown. (d) Line tracings of wound edge at 1-h intervals showing a decrease in migration rates of parental but not CD2AP knockdown cells at 4 and 6 d postconfluency (4 d and 6 d). (e) Line tracings of wound edge at 1-h intervals showing suppression of wound-induced migration of cells expressing exogenous CD2AP at 1 and 2 d postconfluency. (f) Quantitation of migration speed in CD2AP knockdown cells showing a lack of suppression in wound-induced migration after confluency. Representative data from three separate experiments (n = 3) are shown. (g) Actin (phalloidin) staining showing correlation of cell migration with wound-edge protrusions (white arrowheads). Protrusions were suppressed in 4 d and 6 d postconfluent parental cells and cells expressing exogenous CD2AP. (h) Quantitation of migration speed showing suppression of wound-induced migration in 1 d and 2 d postconfluent cells expressing exogenous CD2AP (P < 0.0001). Representative data from three separate experiments (n = 3) are shown.
Figure 10.
Figure 10.
A relationship between actin accumulation at the molecular level, adhesion strengthening at the cellular level, and epithelial cohesion at the tissue level. Actin accumulation at the adherens junction is a balance of actin polymerization, stabilization, and depolymerization. Actin polymerization at the adherens junction requires the coupling of actin nucleation by the arp2/3 complex, actin elongation by EVL, and actin assembly by α-actinin-4. Thus, junction actin assembly is not simply a product of actin polymerization but resulted from the coordinated spatial and temporal coupling between an actin assembly process and a nucleation/polymerization reaction at the adherens junction. After actin is assembled, filament barbed ends are capped by CD2AP. Stabilization of actin by CD2AP contributes to strengthening and maturation of adherens junctions, leading to suppression of cell motility and increase in resilience of epithelial sheet to mechanical stress. Orange boxes represent this work. Red boxes represent work using the same biochemical reconstitution system.

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