Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions

E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells

Mechanism of filopodia initiation by reorganization of a dendritic network

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Λ-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Λ-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Λ-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other

Comparative Dynamics of Retrograde Actin Flow and Focal Adhesions: Formation of Nascent Adhesions Triggers Transition from Fast to Slow Flow

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base

Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

<div><p>The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.</p> </div

Restoration of pp-MRLC organization after blebbistatin washout.

<p>(A–C) Fluorescence microscopy of phalloidin-stained F-actin and immunostained pp-MRLC in cells washed out of 100 µM blebbistatin for 1, 5, or 15 min. Scale bar, 20 µm. Boxed regions are zoomed in the bottom row. (D) Fluorescence intensity profiles of pp-MRLC immunostaining in peripheral regions of cells washed out of 100 µM blebbistatin for 1, 5, or 15 min. Error bars, SD (N = 10 cells, 30 linescans).</p

Blebbistatin inhibits lamellipodia and focal complexes in REF52 fibroblasts.

<p>(A,B) Cell surface topography revealed by platinum replica EM of non-extracted untreated (A) or 100 µM blebbistatin (BS)-treated cells (B). (C,D) Fluorescence microscopy of phalloidin-stained F-actin and immunostained α-actinin in detergent-extracted untreated (C) or 100 µM blebbistatin-treated cells (D). (E) Fractions of the cell perimeter occupied by lamellipodia and ruffles in untreated cells, cells treated with 75 µM or 100 µM blebbistatin, and cells recovering from treatment with 100 µM blebbistatin for indicated periods of time in minutes. Error bars, SD. (F,G) Fluorescence microscopy of phalloidin-stained F-actin and immunostained vinculin in detergent-extracted untreated (F) or 100 µM blebbistatin-treated (G) cells. Boxed regions in C, D, E, and G are zoomed in bottom panels. Scale bars, 2 µm (A,B) and 20 µm (C,D,F,G).</p

EM of cells recovering for 1 min after washout of 100 µM blebbistatin.

<p>(A–C) Correlative fluorescence (C) and EM (A,B) of REF52 cell fluorescently labeled with phalloidin (C, red) and vinculin (cyan) antibody. Focal complexes (cyan spots) colocalize with concave arc-shaped bundles of long actin filaments at the base of lamellipodium (A) or at the cell edge (B). The concave arc in A is continuous with a filopodial bundle terminating at the lamellipodial edge (arrowhead). Panels A and B correspond to boxed regions a and b, respectively, in panel C. (D) Immunogold staining of NMII. Yellow dots mark gold particles that are evenly scattered and not abundant. (E–G) EM of gelsolin-treated cytoskeleton with (E,F) or without (G) NMII immunogold labeling. Boxed region in E is enlarged in F. Yellow dots in E mark NMII immunogold particles. Arrowheads in G point to occasional NMII filaments. Scale bars, 1 µm (A,B),10 µm (C), 0.5 µm (D,F), 1 µm (E) and 200 nm (G).</p

Blebbistatin causes enrichment of pp-MRLC in protrusions.

<p>(A,B) Fluorescence microscopy of phalloidin-stained F-actin and immunostained pp-MRLC in untreated (A) and 100 µM blebbistatin (BS)-treated (B) cells. Boxed regions are zoomed in right panels. Scale bar, 20 µm. (C) Fluorescence intensity profiles of pp-MRLC immunostaining in peripheral regions of untreated or blebbistatin-treated cells. Error bars, SD. (N = 10 cells, 30 linescans). (D) Western blotting with pp-MRLC antibody of total cell lysates (TCL) and pellets of untreated (C) and blebbistatin-treated (BS) cells. Tubulin staining is used as loading control. (E−G) Separation of NMII pools by gradient centrifugation of low speed supernatants from untreated (C, Control) or blebbistatin-treated (BS) cells followed by SDS-PAGE and Western blotting with pp-MRLC antibody. (E) Representative Western blot shows two populations of pp-MRLC with peaks in fractions 3–4 and 7–8 with sedimentation coefficients corresponding to NMII monomers and NMII filaments, respectively. (F) Relative amounts of pp-MRLC in two gradient peaks in untreated (Control) and blebbistatin-treated cells (BS). Cumulative pp-MRLC band intensities in top (#1–5) and bottom (#6–11) gradient fractions are presented as percentage of the total amount of pp-MRLC in respective samples. Error bars, SD (N = 2 experiments). Blebbistatin-treated cell have greater relative amount of pp-MRLC-positive monomers. (G) Average intensities in arbitrary units (a.u.) of pp-MRLC bands in individual fractions plotted against the fraction number. Arrows indicate position of marker proteins: aldolase (7S); catalase (11 S) and ferritin (16S). Error bars, SD (N = 2 experiments).</p

Model for NMII functions during contractile system assembly.

<p>(1) <i>NMII activation</i>: Inactive NMII molecules diffuse to lamellipodia, where they are activated by double phosphorylation of MRLC. (2) <i>Focal complex formation:</i> Active unpolymerized NMII molecules bind actin filaments in lamellipodia and undergo the retrograde flow with them. If two NMII heads bind different actin filaments, one of which is anchored to a nascent adhesion, the resulting strain stabilizes the nascent adhesion and promotes its transformation to a focal complex. Long filaments in filopodia can encounter more NMII molecules increasing a probability of focal complex formation under filopodial bundles. (3) <i>Assembly of NMII filaments:</i> NMII molecules pulling on actin filaments attached to focal complexes experience a greater load, which triggers tension-dependent NMII polymerization at these sites. (4) <i>Stress fiber formation:</i> Multivalent NMII filaments exert large forces sufficient to promote maturation of focal complexes to focal adhesions. They also cross-link and align disordered actin filaments into bundles producing stress fibers. However, additional events, such as recruitment of α-actinin, are needed for the formation of semi-sarcomeric pattern in stress fibers.</p

Dynamics of cell recovery after blebbistatin washout.

<p>(A) Still DIC and fluorescence frames from the time lapse sequence showing a cell cotransfected with mCherry-actin and GFP-MRLC, treated with 100 µM blebbistatin and washed out of the drug for indicated times. Single images from each channel taken before application of blebbistatin are shown in the first column. Bottom row shows merged images. Scale bar, 10 µm. (B) A part of the time lapse sequence of the boxed region in A shown at greater spatial and temporal resolution to illustrate transformation of filopodial roots (4∶30, arrowheads) to a lateral concave arc at the base of lamellipodium (5∶30) and then to more deeply located stress fibers (6∶30–8∶30).</p