Contractile actin cortex is involved in cell morphogenesis, movement, and cytokinesis, but its organization and assembly are poorly understood. During blebbing, the membrane detaches from the cortex and inflates. As expansion ceases, contractile cortex re-assembles under the membrane and drives bleb retraction. This cycle enabled us to measure the temporal sequence of protein recruitment to the membrane during cortex reassembly and to explore dependency relationships. Expanding blebs were devoid of actin, but proteins of the erythrocytic submembranous cytoskeleton were present. When expansion ceased, ezrin was recruited to the membrane first, followed by actin, actin-bundling proteins, and, finally, contractile proteins. Complete assembly of the contractile cortex, which was organized into a cagelike mesh of filaments, took approximately 30 s. Cytochalasin D blocked recruitment of actin and alpha-actinin, but had no effect on membrane association of ankyrin B and ezrin. Ezrin played no role in actin nucleation, but was essential for tethering the membrane to the cortex. The Rho pathway was important for cortex assembly in blebs.

Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously we showed that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. Here we show that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.

The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora B(AIR-2) and the centralspindlin component MKLP1(ZEN-4) causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora B(AIR-2)-inhibited embryos, whereas inhibition of Rac does so in MKLP1(ZEN-4)-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.

Cellular functions, such as division and migration, require cells to undergo robust shape changes. Through their contractility machinery, cells also sense, respond, and adapt to their physical surroundings. In the cytoplasm, the contractility machinery organizes into higher order assemblies termed contractility kits (CKs). Using Dictyostelium discoideum, we previously identified Discoidin I (DscI), a classic secreted lectin, as a CK component through its physical interactions with the actin crosslinker Cortexillin I (CortI) and the scaffolding protein IQGAP2. Here, we find that DscI ensures robust cytokinesis through regulating intracellular components of the contractile machinery. Specifically, DscI is necessary for normal cytokinesis, cortical tension, membrane-cortex connections, and cortical distribution and mechanoresponsiveness of CortI. The dscI deletion mutants also have complex genetic epistatic relationships with CK components, acting as a genetic suppressor of cortI and iqgap1, but as an enhancer of iqgap2. This work underscores the fact that proteins like DiscI contribute in diverse ways to the activities necessary for optimal cell function.

Muscle contraction depends on interactions between actin and myosin filaments organized into sarcomeres, but the mechanism by which actin filaments incorporate into sarcomeres remains unclear. We have found that, during larval development in Caenorhabditis elegans, two members of the actin-assembling formin family, CYK-1 and FHOD-1, are present in striated body wall muscles near or on sarcomere Z lines, where barbed ends of actin filaments are anchored. Depletion of either formin during this period stunted growth of the striated contractile lattice, whereas their simultaneous reduction profoundly diminished lattice size and number of striations per muscle cell. CYK-1 persisted at Z lines in adulthood, and its near complete depletion from adults triggered phenotypes ranging from partial loss of Z line-associated filamentous actin to collapse of the contractile lattice. These results are, to our knowledge, the first genetic evidence implicating sarcomere-associated formins in the in vivo organization of the muscle cytoskeleton.

During cytokinesis, a signal from the central spindle that forms between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375-392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, Mr = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, Mr = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle alpha-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle alpha-actinin. Our results suggest that the 1E12 antigen is a member of the alpha-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Sarcomere maintenance, the continual process of replacement of contractile proteins of the myofilament lattice with newly synthesized proteins, in fully differentiated contractile cells is not well understood. Adenoviral-mediated gene transfer of epitope-tagged tropomyosin (Tm) and troponin I (TnI) into adult cardiac myocytes in vitro along with confocal microscopy was used to examine the incorporation of these newly synthesized proteins into myofilaments of a fully differentiated contractile cell. The expression of epitope-tagged TnI resulted in greater replacement of the endogenous TnI than the replacement of the endogenous Tm with the expressed epitope-tagged Tm suggesting that the rates of myofilament replacement are limited by the turnover of the myofilament bound protein. Interestingly, while TnI was first detected in cardiac sarcomeres along the entire length of the thin filament, the epitope-tagged Tm preferentially replaced Tm at the pointed end of the thin filament. These results support a model for sarcomeric maintenance in fully differentiated cardiac myocytes where (a) as myofilament proteins turnover within the cell they are rapidly exchanged with newly synthesized proteins, and (b) the nature of replacement of myofilament proteins (ordered or stochastic) is protein specific, primarily affected by the structural properties of the myofilament proteins, and may have important functional consequences.

Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.

The cell cortex is essential to maintain animal cell shape, and contractile forces generated within it by nonmuscle myosin II (NMY-2) drive cellular morphogenetic processes such as cytokinesis. The role of actin cross-linking proteins in cortical dynamics is still incompletely understood. Here, we show that the evolutionarily conserved actin bundling/cross-linking protein plastin is instrumental for the generation of potent cortical actomyosin contractility in the Caenorhabditis elegans zygote. PLST-1 was enriched in contractile structures and was required for effective coalescence of NMY-2 filaments into large contractile foci and for long-range coordinated contractility in the cortex. In the absence of PLST-1, polarization was compromised, cytokinesis was delayed or failed, and 50% of embryos died during development. Moreover, mathematical modeling showed that an optimal amount of bundling agents enhanced the ability of a network to contract. We propose that by increasing the connectivity of the F-actin meshwork, plastin enables the cortex to generate stronger and more coordinated forces to accomplish cellular morphogenesis.

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.