Spectrin regulates cell contractility through production and maintenance of actin bundles in the Caenorhabditis elegans spermatheca.
ABSTRACT: Disruption to the contractility of cells, including smooth muscle cells of the cardiovascular system and myoepithelial cells of the glandular epithelium, contributes to the pathophysiology of contractile tissue diseases, including asthma, hypertension, and primary Sjögren's syndrome. Cell contractility is determined by myosin activity and actomyosin network organization and is mediated by hundreds of protein-protein interactions, many directly involving actin. Here we use a candidate RNA interference screen of more than 100 Caenorhabditis elegans genes with predicted actin-binding and regulatory domains to identify genes that contribute to the contractility of the somatic gonad. We identify the spectrin cytoskeleton composed of SPC-1/?-spectrin, UNC-70/?-spectrin, and SMA-1/? heavy-spectrin as required for contractility and actin organization in the myoepithelial cells of the C. elegans spermatheca. We use imaging of fixed and live animals as well as tissue- and developmental-stage-specific disruption of the spectrin cytoskeleton to show that spectrin regulates the production of prominent central actin bundles and is required for maintenance of central actin bundles throughout successive rounds of stretch and contraction. We conclude that the spectrin cytoskeleton contributes to spermathecal contractility by promoting maintenance of the robust actomyosin bundles that drive contraction.
Project description:Stress fibers-contractile actomyosin bundles-are important for cellular force production and adaptation to physical stress and have been well studied within the context of cell migration. However, less is known about actomyosin bundle formation and organization in vivo and in specialized contractile cells, such as smooth muscle and myoepithelial cells. The Caenorhabditis elegans spermatheca is a bag-like organ of 24 myoepithelial cells that houses the sperm and is the site of fertilization. During ovulation, spermathecal cells are stretched by oocyte entry and then coordinately contract to expel the fertilized embryo into the uterus. Here we use four-dimensional confocal microscopy of live animals to observe changes to spermathecal actomyosin network organization during cell stretch and contraction. Oocyte entry is required to trigger cell contraction and concomitant production of parallel actomyosin bundles. Actomyosin bundle size, connectivity, spacing, and orientation are regulated by myosin activity. We conclude that myosin drives actomyosin bundle production and that myosin activity is tightly regulated during ovulation to produce an optimally organized actomyosin network in C. elegans spermathecae.
Project description:The myoepithelial sheath of the Caenorhabditis elegans somatic gonad has non-striated actomyosin networks that provide contractile forces during ovulation, a process in which a mature oocyte is expelled from the ovary. Troponin T and troponin C are known regulators of contraction of the myoepithelial sheath. These are two of the three components of the troponin complex that is generally considered as a striated-muscle-specific regulator of actomyosin contraction. Here, we report identification of troponin I as the third component of the troponin complex that regulates ovulatory contraction of the myoepithelial sheath. C. elegans has four genes encoding troponin-I isoforms. We found that tni-1 and unc-27 (also known as tni-2) encode two major troponin-I isoforms in the myoepithelial sheath. Combination of RNA interference and mutation of tni-1 and unc-27 resulted in loss of the troponin-I protein in the gonad and caused sterility due to defective contraction of the myoepithelial sheath. Troponin-I-depleted gonads were hypercontracted, which is consistent with the function of troponin I as an inhibitor of actomyosin contraction. Troponin I was associated with non-striated actin networks in a tropomyosin-dependent manner. Our results demonstrate that troponin I regulates contraction of non-striated actomyosin networks and is an essential cytoskeletal component of the C. elegans reproductive system.
Project description:The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.
Project description:Actomyosin-based contractility in smooth muscle and nonmuscle cells is regulated by signaling through the small GTPase Rho and by calcium-activated pathways. We use the myoepithelial cells of the Caenorhabditis elegans spermatheca to study the mechanisms of coordinated myosin activation in vivo. Here, we show that redox signaling modulates RHO-1/Rho activity in this contractile tissue. Exogenously added as well as endogenously generated hydrogen peroxide decreases spermathecal contractility by inhibition of RHO-1, which depends on a conserved cysteine in its nucleotide binding site (C20). Further, we identify an endogenous gradient of H2O2 across the spermathecal tissue, which depends on the activity of cytosolic superoxide dismutase, SOD-1. Collectively, we show that SOD-1-mediated H2O2 production regulates the redox environment and fine tunes Rho activity across the spermatheca through oxidation of RHO-1 C20.
Project description:Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction.
Project description:Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/G?s, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/G?s or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/G?s or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 G? subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.
Project description:Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient.
Project description:Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ~190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.
Project description:While the molecular interactions between individual myosin motors and F-actin are well established, the relationship between F-actin organization and actomyosin forces remains poorly understood. Here we explore the accumulation of myosin-induced stresses within a two-dimensional biomimetic model of the disordered actomyosin cytoskeleton, where myosin activity is controlled spatiotemporally using light. By controlling the geometry and the duration of myosin activation, we show that contraction of disordered actin networks is highly cooperative, telescopic with the activation size, and capable of generating non-uniform patterns of mechanical stress. We quantitatively reproduce these collective biomimetic properties using an isotropic active gel model of the actomyosin cytoskeleton, and explore the physical origins of telescopic contractility in disordered networks using agent-based simulations.
Project description:The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone.