Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones.
ABSTRACT: Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase-potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.
Project description:The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.
Project description:Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity. We report that myosin II activity is regulated by PKC during 5-HT responses and that PKC activity is necessary for increases in traction force normally associated with these growth responses. 5-HT simultaneously induces cofilin-dependent decreases in actin network density and PKC-dependent increases in point contact density. These reciprocal effects facilitate increases in traction force production in domains exhibiting decreased actin network density. Interestingly, when PKC activity was up-regulated, 5-HT treatments resulted in myosin II hyperactivation accompanied by catastrophic cofilin-dependent decreases in actin filament density, sudden decreases in traction force, and neurite retraction. These results reveal a synergistic relationship between cofilin and myosin II that is spatiotemporally regulated in the growth cone via mechanocatalytic effects to modulate neurite growth.
Project description:INTRODUCTION:During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKC?/MLC-2). PKC? is activated by growth factor-driven PLC?1 hydrolysis of phosphoinositide bisphosphate (PIP2) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKC? n-terminus mediates PKC? localization to the membrane in relative proximity to PLC?1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKC? mediates the transcellular contractility of fibroblasts. METHODS:To determine PKC? activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKC? constructs were generated; PKC?-CaaX with farnesylation moiety targeting PKC? to the membrane and PKC?-SaaX a non-targeting control. RESULTS:Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKC? (PKC?-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKC? (PKC?-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKC?. CONCLUSION:From these results membrane tethering of PKC? leads to increased force exertion on ECM. Furthermore, our data show PLC?1 regulation of PKC?, at least in part, drives transcellular contractility in fibroblasts.
Project description:Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.
Project description:Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin) network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence length to <1 ?m. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin with a radius of curvature ~300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle and nonmuscle cells.
Project description:Contraction of cortical actomyosin networks driven by myosin activation controls cell shape changes and tissue morphogenesis during animal development. In vitro studies suggest that contractility also depends on the geometrical organization of actin filaments. Here we analyze the function of actomyosin network topology in vivo using optogenetic stimulation of myosin-II in Drosophila embryos. We show that early during cellularization, hexagonally arrayed actomyosin fibers are resilient to myosin-II activation. Actomyosin fibers then acquire a ring-like conformation and become contractile and sensitive to myosin-II. This transition is controlled by Bottleneck, a Drosophila unique protein expressed for only a short time during early cellularization, which we show regulates actin bundling. In addition, it requires two opposing actin cross-linkers, Filamin and Fimbrin. Filamin acts synergistically with Bottleneck to facilitate hexagonal patterning, while Fimbrin controls remodeling of the hexagonal network into contractile rings. Thus, actin cross-linking regulates the spatio-temporal organization of actomyosin contraction in vivo, which is critical for tissue morphogenesis.
Project description:Actomyosin networks generate contractile force that changes cell and tissue shape. In muscle cells, actin filaments and myosin II appear in a polarized structure called a sarcomere, in which myosin II is localized in the center. Nonmuscle cortical actomyosin networks are thought to contract when nonmuscle myosin II (myosin) is activated throughout a mixed-polarity actin network. Here, we identified a mutant version of the myosin-activating kinase, ROCK, that localizes diffusely, rather than centrally, in epithelial cell apices. Surprisingly, this mutant inhibits constriction, suggesting that centrally localized apical ROCK/myosin activity promotes contraction. We determined actin cytoskeletal polarity by developing a barbed end incorporation assay for Drosophila embryos, which revealed barbed end enrichment at junctions. Our results demonstrate that epithelial cells contract with a spatially organized apical actomyosin cortex, involving a polarized actin cytoskeleton and centrally positioned myosin, with cell-scale order that resembles a muscle sarcomere.
Project description:Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.
Project description:KEY POINTS:Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease. ABSTRACT:Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.
Project description:Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.