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First published online 25 October 2005
doi: 10.1242/jcs.02626

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Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network

Department of Biological Sciences, 4249 Fifth Avenue, Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, USA

View larger version (110K): [in a new window]   Fig. 1. Shrm is localized to the apical junctional complex (AJC). (A) Cryosections from e9.25 embryos were stained to detect Shrm (green) and ß-catenin (red). Shrm is distributed in an apically positioned honeycomb array in a projection of confocal sections. ap, apical. (B) An enlarged single optical section from the boxed region in panel A. Arrows indicate laterally positioned ß-catenin (red) and white arrowheads indicate apically positioned Shrm (green). (C) Cryosections from the truck region of an e9.5 embryo stained to detect Shrm (green, C'), ß-catenin (red) and F-actin (blue, C''). White arrowheads indicate apically positioned Shrm and F-actin. (D) Cryosections of e9.5 mouse embryos stained to detect Shrm (green) and Nectin-3 (red). A projected image shows Shrm (green) and Nectin-3 (red) in a honeycomb array. A single optical section demonstrating the overlapping distribution of Shrm and Nectin-3 in the putative AJC (white arrowhead) is shown in D'. (E) Equal amounts (30 µg) of total cell lysate from induced and un-induced ShrmL cells were assayed by western blot analysis using anti-Shrm sera. Filters were re-probed to detect ß-catenin to verify equal protein loading. (F and G) ShrmL cells were grown on Transwell filters for 48 hours in the presence (F) or absence (G) of doxycyclin and stained to detect Shrm (green) and ZO-1 (red). Top panels are X-Y projections and bottom panels are X-Z projections. Arrows indicate cells with rounded and polyhedral morphology. (H,I) ShrmL cells plated on Transwell filters were grown in the presence (H) or absence (I) of doxycyclin for 48 hours and stained to detect E-cadherin. Top panels are projections in the X-Y dimension and bottom panels are X-Z projections. Panels I' and I'' show individual optical sections of E-cadherin staining at the level of the basal surface and the AJC, respectively. Asterisks, constricted cells; dots, stretched cells. In all panels, scale bar is 15 µm, open arrowheads indicate the position at which X-Z projections were generated; ap, apical.

View larger version (113K): [in a new window]   Fig. 2. Shrm controls apical morphology in MDCK cells. (A-C) A mixture of T23 and ShrmL cells were grown on Transwell filters in the absence of doxycyclin and stained to detect Shrm (green), ZO-1 (red) and E-cadherin (B,C). Asterisks indicate constricted, ShrmL-expressing cells and dots indicate stretched, non-expressing cells. Panels B and C show single optical sections of E-cadherin staining at the basal and apical surfaces, respectively. (D) Scatter plot of apical (ap) and basal (bsl) areas of ShrmL expressing (1), neighboring wild-type (2) and non-neighboring, wild-type (3) cells in mixed populations of ShrmL expressing and wild-type T23 cells. Each point represents one cell. (E-G) Clusters of increasing numbers of ShrmL-expressing cells (stained to detect Shrm) show the configuration of cells at the level of the AJC. (H-J) Cysts generated from control cells (H) or induced ShrmL cells (I and J) were stained to detect Shrm (green), ZO-1 (red) and F-actin (blue). Arrows denoted cells with constricted apical surfaces. Scale bar represents 15 µm in all panels.

View larger version (110K): [in a new window]   Fig. 3. Apical targeting of the C-terminus of Shrm alters cell morphology. (A) Schematic of the chimeric protein consisting of Endolyn and the C-terminus of Shrm (Endo-ShrmC) and the control chimeric protein consisting of Endolyn and the C-terminus of Shrm truncated to remove ASD2 (Endo-ShrmC). (B,C) T23 MDCK cells on Transwell filters were transiently transfected with expression vectors for Endo-ShrmC (B) or Endo-ShrmC (C) and stained to detect Shrm (green) and ZO-1 (red). (D) Equal amounts (30 µg) of total cell lysate from induced and un-induced T23-Endo-ShrmC cells were assayed by western blot analysis using anti-Shrm sera. Filters were re-probed to detect ß-catenin to verify equal protein loading. (E,F) Endo-ShrmC cells were grown on Transwell filters in the presence (E) or absence (F) of doxycyclin and stained to detect ZO-1 (green, E' and F'), E-cadherin (red, E'' and F''), or Shrm (blue, E''' and F'''). Boxed regions in (F) and (F') are enlarged in the insets. Arrows denote finger-like structures. (G,H) Lateral (G' and H') and apical (G'' and H'') optical sections from the un-induced (G' and G'') and induced (H' and H'') cells shown in panels E and F, respectively. The lateral sections are from the mid-way point of the apical-basal axis and the sub-apical sections are taken at the level of the AJC as defined by ZO-1 staining. In all panels, scale bar represents 15 µm. Open arrowheads denote position of the X-Z projections shown beneath relevant panels.

View larger version (166K): [in a new window]   Fig. 4. Shrm-induced changes in morphology are associated with alterations in the organization of the apical actin cytoskeleton. (A-C) Control (A), induced ShrmL (B), or induced Endo-ShrmC (C) cells were grown on filters and stained to detect F-actin (A-C, A'-C') and ZO-1 (A''-C''). Boxed regions in A-C are enlarged in A'-C' and A''-C''. Inset in (C) shows Endo-ShrmC expression. Arrowhead denotes actin rings and arrows denote apical actin fibers. Images are projections of 0.2 µm sections spanning the AJC and apical plasma membrane. In all panels, scale bar represents 15 µm.

View larger version (140K): [in a new window]   Fig. 5. The activity of myosin II and Rock are required for Shrm-induced cell shape changes. (A,B) Control cells were treated with blebbistatin (bleb) or Y27632 and stained to detect ZO-1. (C-H) Induced ShrmL cells (C,E,G) or Endo-ShrmC cells (D,F,H) were treated with DMSO (C and D), 100 µM blebbistatin (E and F), or 10 µM Y-27632 (G,H) and stained to detect Shrm (green) and ZO-1 (red). Arrowheads denote positions of the X-Z projections shown beneath each panel. (I-K) Control cells (I) or ShrmL-expressing cells (J and K) were transfected with expression vectors for either GFP-RhoA (I and J) or GFP-RhoA19N (K) and stained to detect GFP, Shrm and E-cadherin. Merged images (I-K) show GFP (green) and ShrmL (red). Individual fluorescent signals for GFP (I'-K') and E-cadherin (I''-K'') are included. Arrows indicate transfected cells. In all panels, scale bar represents 15 µm.

View larger version (136K): [in a new window]   Fig. 6. The role of F-actin in Shrm-induced constriction. (A-D) Cells expressing ShrmL (A and B) or Endo-ShrmC cells (C and D) were treated with DMSO (A and C) or 1 µM cytochalasin D (B and D) for 30 minutes and stained to detect ZO-1. (E,F) Wild-type T23 cells grown on Transwell filters were treated with DMSO (E' and E'') or 2 µM CD (F' and F'') for 30 minutes, stained with phalloidin to detect F-actin and imaged by confocal microscopy to visualize the cytoskeleton at either the apical or basal surface. (G-I) Control (G), ShrmL expressing (H), or Endo-ShrmC expressing (I) cells were treated with 2 µM cytochalasin D for 30 minutes and stained to detect E-cadherin (green), ZO-1 (red, H' and I'), or Shrm (blue, H'' and I''). (J-L) Control (J), ShrmL expressing (K), or Endo-ShrmC expressing (L) cells were pre-treated with 100 µM blebbistatin for 30 minutes followed by 2 µM cytochalasin D for 30 minutes and stained to detect E-cadherin (green), ZO-1 (red, K' and L'), or Shrm (blue, K'' and L''). Scale bar represents 15 µm in all panels.

View larger version (142K): [in a new window]   Fig. 7. Shrm alters the distribution of myosin II in MDCK cells. (A-C) Control (A), ShrmL-expressing (B) and Endo-ShrmC-expressing (C) cells were grown on filters and stained to detect non-muscle myosin II-B (green, A'-C', A'''-C''') and ZO-1 (red, A''-C''). Images are projections of 0.2 µm optical sections that span the AJC and apical plasma membrane. Areas boxed in A'-C' are shown enlarged in A'''-C'''. Lower panels represent X-Z projections of the entire apical-basal axis. Arrowheads denote where X-Z sections were generated. Arrows denote the regularly spaced distribution of non-muscle myosin II-B. (D,E) Mixed populations of wild-type T23 cells and ShrmL cells were grown on filters in the absence of doxycyclin and stained to detect myosin II-B (green, D' and E') and ZO-1 (red) or myosin IIB, ZO-1 and ShrmL (blue, E''). (F) Induced ShrmL cells were stained to detect myosin II-B (green) and F-actin (red). Arrowhead denotes position of X-Z projection shown beneath panel. F' and F'' show myosin llB and F-actin signals alone. (G) Induced ShrmL cells were stained to detect myosin IIB (green, G'), ShrmL (red, G'') and F-actin (blue, G'''). (H) Induced ShrmL cells grown on coverslips were stained to detect pp-RMLC (green, H'), ZO-1 (red, H'') and F-actin (blue, H'''). (I) Induced ShrmL cells were transiently transfected with a GFP--Actinin expressing vector and stained to detect myosin II-B (red, I') and GFP (green, I''). Scale bar equals 15 µm in A-H and 1.5 µm in I.

View larger version (90K): [in a new window]   Fig. 8. Shrm is required for proper myosin IIB distribution during neural tube closure. (A-D) Cryosections of wild type (A and B) or shrm mutant (C and D) e9.25 mouse embryos were stained to detect non-muscle myosin II-B (green, A'-D') and ß-catenin (red). Panels A and C show sections through the cranial neural folds while B and D are sections through the trunk neural tube. Scale bar equals 15 µm, ap, apical surface. (E) Model of Shrm function in neural tube closure. Shown are schematics of the neural tube in both cranial and trunk regions indicating the presence of bending (a) and non-bending (b) regions of the cranial and trunk neural tube. Cells in both types of regions contain contractile rings (c) in the AJC that provide force around the circumference of the cell to mediate either apical constriction or provide tension. (d) Shrm is recruited to the AJC via either F-actin that is already localized there or another protein in the AJC (double head arrows). Once in the AJC complex, the C-terminal domain of Shrm facilitates the assembly of a contractile actomyosin complex (green actin and myosin). The C-terminus of Shrm could act directly or through a second cellular protein (?) to facilitate formation of the complex. It is predicted that Rock could be needed in parallel to maintain a population of active myosin II.

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