January 17, 2012;
A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration.
Collective cell migration requires maintenance of adhesive contacts between adjacent cells, coordination of polarized cell protrusions, and generation of propulsive traction forces. We demonstrate that mechanical force applied locally to C-cadherins on single Xenopus mesendoderm
cells is sufficient to induce polarized cell protrusion and persistent migration typical of individual cells within a collectively migrating tissue
. Local tension on cadherin adhesions induces reorganization of the keratin intermediate filament network toward these stressed sites. Plakoglobin
, a member of the catenin family, is localized to cadherin adhesions under tension and is required for both mechanoresponsive cell behavior and assembly of the keratin cytoskeleton at the rear of these cells. Local tugging forces on cadherins occur in vivo through interactions with neighboring cells, and these forces result in coordinate changes in cell protrusive behavior. Thus, cadherin-dependent force-inducible regulation of cell polarity in single mesendoderm
cells represents an emergent property of the intact tissue
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References [+] :
Figure 1. Force Application to Cadherin Induces Oriented Monopolar Protrusive Behavior(A) SEM of mesendoderm (blue shading) from dorsal region of Xenopus gastrula with overlying blastocoel roof and attached FN matrix removed reveals basal surfaces of the mesendoderm cells with underlapping monopolar lamelliform protrusions (white arrowheads) oriented in the direction of travel (arrow). A transitional group of nonpolar cells (green shading) separates mesendoderm and mediolaterally intercalating mesoderm (yellow shading). Note that the long axis of each mesendoderm cell (i.e., in direction of travel) is oriented perpendicular to that of the mediolaterally intercalating mesoderm cells.(B) Schematic of experimental strategy for magnetic bead pull assay (see Experimental Procedures for details).(C) Still images from time-lapse movie (Movie S1) of a single multipolar mesendoderm cell plated on FN.(D) Still images from time-lapse movie (Movie S2) of an isolated mesendoderm cell, plated on FN and with C-cadFc coated bead attached (arrowhead).(E) Still images from time-lapse movie (Movie S2). Same cell as (D), C-cadFc bead pulled by magnet indicated at right (red magnet icon). A lamellipodium forms (arrow) opposite the direction of bead pull and results in directed cell migration.(F) Quantitation of protrusion angles relative to cell centroid (center of rose diagram) and magnet at right (0. y axis for rose diagram represents percent of total protrusions.(G) Quantitation of protrusions per cell after bead attachment and pull.Data are represented as mean SEM. All scale bars, 50 μm. (C) Times shown in minutes:seconds. See also Figures S1 and S2, and mmc2VIDEO, mmc3VIDEO and mmc4VIDEO.
Figure 2. Keratin Organization Is Regulated by Tension on Cell-Cell Contacts(A) Single cell on FN, labeled with Alexa555-dextran (red) and expressing GFP-XCK1(8) to visualize KIFs (green).(B) Pair of fixed mesendoderm cells immunostained for C-cadherin (red) and XCK1(8) (green). Dashed line, cell-cell boundary.(C) Cell within mesendoderm tissue explant on FN labeled with Alexa555-dextran (red) and expressing GFP-XCK1(8) (green).(D) Sagittal perspective of mesendoderm cell in bisected embryo immunostained for C-cadherin (red) and XCK1(8) (green). KIFs in posterior of polarized cells (arrowheads in B) and along tissue leading edge (arrow in C).(E and E′) Single mesendoderm cell on FN labeled with Alexa555-dextran (red), expressing GFP-XCK1(8) (green). C-cadFc bead (dashed circle) attached to cell (E) and then pulled for 20 min (E′). Arrows, leading edge protrusion.(F) Brightfield image of cell pair on FN, polarized in opposing directions (double arrow).(G and H) Cell pairs expressing GFP-XCK1(8), plated on FN (G) or PLL (H). Dashed line, cell-cell boundary. Cell borders outlined by dotted line in (G). All scale bars, 25 μm. See also Movie S4.
Figure 3. Keratin and PG Are Required for Polarized Protrusive Behaviors(A) Quantitation of protrusion angles from XCK1(8) morphant cells with C-cadFc beads attached and following bead pull. See also Figure S3 and Movie S5.(B) GAP43-GFP labels plasma membranes in intact mesendoderm explants prepared from control morphant (left) and XCK1(8) morphant embryos (right). Green arrowheads indicate protrusions in the direction of tissue movement and red arrowheads mark protrusions in any other direction. See also Movie S6.(C) Quantitation of protrusion angles from PG morpholino knockdown cells with C-cadFc beads attached and following bead pull. See also Figure S4 and Movie S7.(D) Quantitation of protrusion number per cell in normal and PG morphant cells. Data are represented as mean SEM.(E and F) Quantitation of protrusion angles, where 180equals direction of tissue migration, in control morphant explants (E) and PG morphant explants (F). Leading cells = row 1, following cells = rows 2-4. In panels at right, GAP43-GFP labels plasma membrane of mesendoderm explants from control morphant and PG morphant embryos. See also Movie S8. Green arrowheads indicate protrusions in the expected direction of tissue movement and red arrowheads mark protrusions in any other direction. All scale bars, 25 μm.
Figure 4. Recruitment of PG to Stressed Cadherin Adhesions(A and A′) 3D rendered side view of a normal cell injected with Alexa555-dextran (red) and expressing PG-GFP (green) before (A) and after (A′) C-cadFc bead pull. Location of bead, dashed circle.(B′) Cells expressing either PG-GFP (B and B′) or C-cadherin-GFP (C and C′), plated on either FN (B and C) or PLL (B′ and C′) and allowed to form cohesive pairs. Arrowheads indicate plane of cell-cell boundaries.(D) Mesendoderm cells in live tissue expressing PG-GFP (red), mCherry-XCK1(8) (green), and labeled with Alexa647-dextran (gray). Image is a collapsed 2 μm Z-stack of the posterior-lateral region of two adjacent cells in a mesendoderm explant. Outlined region in (D) is shown in independent color channels of plakoglobin-GFP (D′), mCherry-XCK1(8) (D″), and dextran (D″′).(E) C-cadherin and PG were immunoprecipitated from whole embryo extracts and immunoblotted as indicated. α5 integrin immunoprecipitates served as negative controls. All scale bars, 15 μm.
Figure 5. Requirement of PG for Cadherin/Keratin Association(A and B) Single cells labeled with Alexa-dextran, expressing GFP-XCK1(8) (green) and plated on FN. (A) and (A′) show a normal cell (blue dextran), and (B) and (B′) show a PG morphant cell (magenta dextran). C-cadFc bead (circle) bound (A and B), then pulled (A′ and B′).(C and D) Control morphant (blue dextran) (C) and PG morphant (magenta dextran) (D) mesendoderm tissue explants expressing GFP-XCK1(8) (green). See also Movie S8 and Figure S5.(E and F) Control (E) and PG morphant (F) mesendoderm in whole embryos immunostained for XCK1(8) (green) and β-catenin (red). (C) Arrows, cabling along anterior of leading edge cells. Arrowheads, KIF aggregation near cell-cell contacts. All scale bars, 25 μm.(G) Embryos were injected with XCK1(8)-GFP, with or without PG morpholino. (G) Immunoblots of embryo lysates show expression levels of XCK1(8)-GFP and endogenous PG with or without PG morpholino (PG-MO). (H) C-cadherin immunoprecipitates immunoblotted for XCK1(8)-GFP and C-cadherin with or without PG-MO. (I) Quantitation of three independent coimmunoprecipitation experiments shown as mean SEM.
Figure 6. Requirement for PG and Keratin in Normal Mesendoderm In VivoScanning electron micrographs of Xenopus embryos from which the overlying blastocoel roof was removed to reveal the basal aspect of the underlying mesendoderm (as in Figure 1A). Leading edge mesendoderm cells and direction of migration in all images is toward top. Images were acquired of (A and D) control morpholino-injected embryos, (B and E) PG morpholino-injected embryos, and (C and F) XCK1(8) morpholino-injected embryos. En face view of basal aspect shown in (A)C) and oblique view of the basal surface shown in (D)F). Arrowheads indicate a sampling of cell protrusions. Scale bars, 50 μm.
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