Cell-cell contact modulation of myosin organization in the early mouse embryo

Preimplantation mouse embryos are characterized by a polarized distribution of cortical myosin (J. S. Sobel (1983). Dev. Biol. 95, 227-231.). Myosin was present in the peripheral regions of the blastomers and was not detectable in regions of cell contact. Disaggregation of the embryos yielded blastomeres which had a continuous layer of cortical myosin. Development of new contact relations in aggregates, between daughter cells of divided blastomeres, and in chimaeras resulted in renewed polarization of cortical myosin. The results indicate that continuous cell contact interaction modulates the distribution of myosin throughout the preimplantation stages of development. The loss of detectable myosin from regions of cell contact was correlated with development of cell contacts that remained stable after Triton X-100 extraction.     

Extracellular matrix- and cytoskeleton-dependent changes in cell shape and stiffness

Cell spreading is correlated with changes in important cell functions including DNA synthesis, motility, and differentiation. Spreading is accompanied by a complex reorganization of the cytoskeleton that can be related to changes in cell stiffness. While cytoskeletal organization and the resulting cell stiffness have been studied in motile cells such as fibroblasts, less is known of these events in nonmigratory, epithelial cells. Hence, we examined the relationship between cell function, spreading, and stiffness, as measured by atomic force microscopy. Cell stiffness increased with spreading on a high density of fibronectin (1000 ng/cm(2)) but remained low in cells that stayed rounded on a low fibronectin density (1 ng/cm(2)). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting f-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrate the relationship between cell stiffness and spreading in hepatocytes. They specifically show that normal actin and myosin function is required for hepatocyte spreading and DNA synthesis and demonstrate opposing effects on cell stiffness upon disruption of actin and myosin.     

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges. Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.     

Two-step positioning of a cleavage furrow by cortexillin and myosin II

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.     

Myosin is involved in postmitotic cell spreading

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.     

Membrane-cytoskeletal interactions in the early mouse embryo

Differentiation in the early mouse embryo begins at the 8-cell stage when the blastomeres flatten against each other by active spreading movements and surface and cytoplasmic elements become concentrated in the apical (uncontacted) region of the cells. A ring of cortical myosin marks the demarcation between the contacted and the uncontacted cellular domains. The organization of the cortical contractile apparatus in the blastomeres bears a formal resemblance to that of other cells that are engaged in similar motile activities. It has been proposed that a flow of cortical filaments could provide the motor that powers these movements. The applicability of such a cortical flow model to the early embryo and the implications for cell flattening and cell polarization are discussed in this review.     

A role for integrin in the formation of sarcomeric cytoarchitecture

We propose that integrins help to coordinate the differentiation of the internal, sarcomeric cytoarchitecture of a muscle fiber with its immediate environment and are essential for correct integration of muscle cells into tissue. We found that integrin alpha PS2 beta PS accumulated at contact regions of Drosophila embryo cells cultured in D-22 medium on Drosophila laminin. Myotubes formed, but subsequent addition of serum or fibronectin was needed for sarcomere formation: integrin and actin became concentrated at Z-bands; myosin and actin occurred between the Z-bands. This change failed to occur in the multinucleate myotubes derived from integrin beta PS null myospheroid mutants. In normal embryos/early larvae, integrin was located at Z-bands and at muscle insertions. Myogenesis and Z-bands were defective in myospheroid embryos. Attachment, spreading, and growth of myoblasts and neurons on the laminin substrate utilized different binding proteins and were independent of integrin.     

Non-sarcomeric mode of myosin II organization in the fibroblast lamellum

The organization of myosin in the fibroblast lamellum was studied by correlative fluorescence and electron microscopy after a novel procedure to reveal its underlying morphology. An X-rhodamine analog of conventional smooth muscle myosin (myosin II) that colocalized after microinjection with endogenous myosin was used to trace myosin distribution in living fibroblasts. Then, the same cells were examined by EM of platinum replicas. To visualize the structural arrangement of myosin, other cytoskeletal fibrillar structures had to be removed: microtubules were depolymerized by nocodazole treatment of the living cells before injection of myosin; continued nocodazole treatment also induced the intermediate filaments to concentrate near the nucleus, thus removing them from the lamellar region; actin filaments were removed after lysis of the cells by incubation of the cytoskeletons with recombinant gelsolin. Possible changes in myosin organization caused by this treatment were examined by fluorescence microscopy. No significant differences in myosin distribution patterns between nocodazole-treated and control cells were observed. Cell lysis and depletion of actin also did not induce reorganization of myosin as was shown by direct comparison of myosin distribution in the same cells in the living state and after gelsolin treatment. EM of the well-spread, peripheral regions of actin-depleted cytoskeletons revealed a network of bipolar myosin mini-filaments, contracting each other at their terminal, globular regions. The morphology of this network corresponded well to the myosin distribution observed by fluorescence microscopy. A novel mechanism of cell contraction by folding of the myosin filament network is proposed.     

Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading

The actomyosin cytoskeleton plays prominent roles in cell spreading and migration. To address the roles of myosin II isoforms and to estimate the region where the myosin IIs are activated in spreading cells, we examined the immunolocalization of myosin II isoforms and phosphorylated RLCs in the spreading MRC-5 cells. We observed the formation of actin ring-like structure at the base of the lamella. Both myosin IIA and IIB were predominantly localized there. Myosin IIA and diphosphorylated RLC were distributed outside of the region where myosin IIB and monophosphoryated RLC were distributed predominantly. Inhibition of Rho-kinase resulted in the disappearance of the diphosphorylation of RLC, moreover, it accelerated the rate of cell spreading and induced an aberrant cell shape at later stage of spreading. These results indicate that diphosphorylation of RLCs of myosin IIA by Rho-kinase in lamella is responsible for the cell to spread properly.     

Distribution of actin and myosin in mouse trophoblast: Correlation with changes in invasiveness during development in vitro

Mouse trophoblast is an invasive tissue that undergoes conversion to a noninvasive state during normal development. We examined the distribution of actin and myosin during trophoblast development in vitro with double label fluorescence microscopy using fluoresceinated subfragment-1 of myosin to identify actin and indirect immunofluorescence with rhodamine-conjugated antibody to detect myosin. During the outgrowth stage trophoblast spread as a sheet by active movement of the marginal cells. These cells exhibited different patterns of actin and myosin distribution in connection with lamellar extension and fiber formation. Marginal and submarginal cells were packed with overlapping layers of actin fibers, some of which were organized into a lattice that extended throughout the trophoblast. The cytoskeletal function of the fibers appeared to involve maintenance of the cells in a coherent sheet. Cessation of trophoblast spreading was associated with conversion of the cell sheet into a cell network. Cells stained more densely for actin and myosin and contained distinctive actomyosin condensations in the cortex and the cytoplasm. At the same time there was disorganization and then loss of the actin fiber system. These changes in actin and myosin distribution may be associated with mechanisms that control invasiveness by limiting trophoblast expansion.     

Macrophages form circular zones of very close apposition to IgG-coated surfaces

When phagocytes spread on surfaces coated with ligands such as IgG, they form a tight seal with the substrate. This seal excludes soluble macromolecules in the medium from the interface between the cell and substrate. In contrast, when cells spread on control surfaces that are not coated with ligands, the underside of the cell remains freely accessible to soluble proteins (Wright and Silverstein: Nature 309:359, 1984). We employed reflection-interference microscopy (RIM) to determine where the seal forms during interaction with ligand (IgG)-coated surfaces. Human monocyte-derived macrophages (MO) were plated at 37 degrees C on dinitrophenylated (DNP)-glass coverslips (control substrate), IgM anti-DNP-DNP-coated glass (control substrate), or on IgG anti-DNP-DNP-coated glass (phagocytosis-promoting substrate). Live or fixed cells were examined by RIM. Spreading on control surfaces at 37 degrees C was complete in 25 minutes, whereas spreading on IgG-coated surfaces was maximal within 15 minutes and resulted in cell-substrate contact area 1.6 X that of control cells. Within 1 h at 37 degrees C, 90% of MO that spread on IgG-coated substrates, but not on control substrates, excluded macromolecules from their underside. A minor population of cells (19%) exhibited a uniform iron gray RIM appearance indicating an even, close approach to the substrate. These cells may represent early stages of frustrated phagocytosis. In contrast to cells on control substrates, 70% of cells on IgG-coated substrates developed continuous peripheral dark rings in RIM indicative of close association with the substrate. Essentially all cells with peripheral dark rings in RIM excluded macromolecules from their underside. Enclosed within this ring was an area of greater separation between the cell membrane and the substrate, as indicated by the lighter grey of this region in RIM and by the accessibility of substrate to anti-substrate antibody when breaks in the dark ring occur. Thus, MO can create a closed compartment between plasma membrane and substrate that excludes proteins in the surrounding medium, thereby protecting substances secreted into this space from potentially inhibitory substances in the medium.     

Drosophila myosin phosphatase and its role in dorsal closure

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.     

Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum : Further evidence for the polar growth of cytoplasmic microtubules in vivo

Mouse 3T3 cells were allowed to attach to and spread on glass. The expression of cytoplasmic microtubules during the respreading process was monitored by immunofluorescence microscopy using monospecific antibody against tubulin. During radial attachment of the cells a ring of flattened cytoplasm is seen around the nucleus. Cytoplasmic microtubules then enter this spreading ring from the perinuclear region and elongate toward the plasma membrane. At later times microtubules appear perpendicular to the plasma membrane and seem to be in intimate contact with it giving the impression that they “stretch” the cytoplasm. When the cells assume their typical fibroblastic shape numerous microtubules are seen. They traverse the cytoplasm. Some come close to the plasma membrane and some bend to conform to the shape of the cell. Changes in microtubular organization correlate well with changes in cell shape. These results together with our previous observations on the assembly of cytoplasmic microtubules upon recovery from colcemid treatment suggest that microtubules may grow as polar structures from a microtubular organizing center towards the plasma membrane. The hypothesis that cytoplasmic microtubules might confer polarity on the cell is discussed.     

The effects of cell-cell contact on the spreading of pigmented retina epithelial cells in culture

The behaviour of chick embryo pigmented retina epithelial (PRE) cells has been studied in living and fixed cultures. Isolated PRE cells lacking contacts with other cells were characteristically only poorly spread upon the substrate, blebbed vigorously and lacked leading lamellae. PRE cells incorporated into islands or sheets of cells were extensively spread upon the substrate, lacked blebs and displayed typical leading lamellae if marginally positioned in an island. Observations of living cultures demonstrated that within 3 h of establishing contact with an island of cells a previously isolated PRE cell lost the morphology characteristic of isolated cells and became indistinguishable from its neighbours in the island. Measurements of the area of substrate occupied by single cells and cells in 2-cell islands suggests that similar changes occur as two cells make contact to form a 2-cell island. The evidence suggests that these changes are a direct response to the establishment of a cell-cell contact and I propose that the phenomenon be termed ‘contact-induced spreading’.Contact-induced spreading is not an ‘all or none’ phenomenon since isolated PRE cells can spread extensively and cease blebbing in the absence of cell contact. However a given isolated PRE cell spends only a very small proportion of its time displaying this well spread morphology and therefore at any time the majority of isolated PRE cells display the poorly spread morphology.The possible relationship between contact-induced spreading and other cellular interactions known to be dependent on cell-cell contact is discussed.     

Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis

BACKGROUND: After anaphase, the segregated chromosomes are sequestered by cytokinesis into two separate daughter cells by a cleavage furrow formed by the actomyosin-based contractile ring. The failure to properly position the contractile ring between the segregated chromosomes can result in aneuploidy. In both C. elegans embryos and human cells, the central spindle regulates division-plane positioning in parallel with a second pathway that involves astral microtubules. RESULTS: We combined genetic and pharmacological manipulations with live cell imaging to spatially separate the two division cues in a single cell. We demonstrate that the two pathways for furrow formation are mechanistically and genetically distinct. By following the distribution of green fluorescent protein (GFP)-tagged nonmuscle myosin, we have found that the astral pathway for furrow formation involves the negative regulation of cortical myosin recruitment. An asymmetrically positioned spindle induces the asymmetric cortical accumulation of myosin. This cortical myosin behaves as a coherent contractile network. If the cortical network is nonuniform over the cell, the cortical contractile elements coalesce into a single furrow. This coalescence requires interconnections among contractile elements. CONCLUSIONS: We conclude that the two pathways of cleavage-furrow formation are mechanistically distinct. In particular, we conclude that the astral pathway for cleavage-furrow formation involves the negative regulation of myosin distribution by astral cues.     

Functional analysis of the Drosophila diaphanous FH protein in early embryonic development

The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.     

Analysis of cell division using fluorescently labeled actin and myosin in living PtK2 cells

Actin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.     

Organization of non-muscle myosin during early murine embryonic differentiation

A monoclonal antibody (3D10) recognizing myosin heavy chain was isolated following immunization with a synthetic peptide sequence of eight amino acids. The antibody reacted with purified rabbit skeletal myosin and light mero-myosin in enzyme-linked immunosorbent assays and Western immunoblotting. A band of approximately 200 kDa was detected in cell extracts of an embryonal carcinoma (EC) cell line (P19EC) and one of its cloned differentiated derivatives, suggesting reactivity against non-muscle myosin. By indirect immunofluorescence, typical myosin banding patterns were observed in cryostat sections of human skeletal and cardiac muscle tissue. In undifferentiated P19EC cells, speckled immunofluorescent staining was observed in the cytoplasm that became organized in cortical rings where the cells made direct contact with each other. These rings consisted of circular bundles of F-actin decorated by myosin. Undifferentiated embryonic stem (ES) cells derived directly from mouse embryos shared the same features, although the pattern was less pronounced. Human testicular primary germ cell tumours showed cortical staining in the embryonal carcinoma component reminiscent of the staining of EC cells in vitro while cytoplasmic staining was observed in tumour cells with a differentiated morphology. In preimplantation embryos, the immunofluorescent staining was observed at cell apices of blastomeres of morula stage embryos. In blastocysts, staining of inner cell mass cells was not detectable. By contrast, various differentiated derivatives of P19EC contained extensive F-actin microfilament bundles throughout the cytoplasm decorated with myosin. Thick stress fibers in filopodious extensions of cells were particularly highly decorated by myosin. Over the nucleus, linear arrays of myosin containing speckled patterns of immunofluorescence were observed that were not associated with F-actin. The same pattern of staining could be observed in trophectoderm cells of the blastocyst. We conclude that embryonic non-muscle myosin is organized in specific patterns depending on the state of differentiation. As the myosin is primarily associated with F-actin we suspect that it forms part of a contractile apparatus that may have significance during embryonic development.     

Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells

We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.