Yixin Ren

Interactions between Myosin and Actin Crosslinkers Control Cytokinesis Contractility Dynamics and Mechanics

INTRODUCTION Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium. RESULTS We establish that the actin polymers are assembled into a meshwork and that myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. We show that myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, we show that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics. CONCLUSIONS These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.

Mechanosensing through Cooperative Interactions between Myosin II and the Actin Crosslinker Cortexillin I

BACKGROUND Mechanosensing governs many processes from molecular to organismal levels, including during cytokinesis where it ensures successful and symmetrical cell division. Although many proteins are now known to be force sensitive, myosin motors with their ATPase activity and force-sensitive mechanical steps are well poised to facilitate cellular mechanosensing. For a myosin motor to experience tension, the actin filament must also be anchored. RESULTS Here, we find a cooperative relationship between myosin II and the actin crosslinker cortexillin I where both proteins are essential for cellular mechanosensory responses. Although many functions of cortexillin I and myosin II are dispensable for cytokinesis, all are required for full mechanosensing. Our analysis demonstrates that this mechanosensor has three critical elements: the myosin motor where the lever arm acts as a force amplifier, a force-sensitive bipolar thick-filament assembly, and a long-lived actin crosslinker, which anchors the actin filament so that the motor may experience tension. We also demonstrate that a Rac small GTPase inhibits this mechanosensory module during interphase, allowing the module to be primarily active during cytokinesis. CONCLUSIONS Overall, myosin II and cortexillin I define a cellular-scale mechanosensor that controls cell shape during cytokinesis. This system is exquisitely tuned through the enzymatic properties of the myosin motor, its lever arm length, and bipolar thick-filament assembly dynamics. The system also requires cortexillin I to stably anchor the actin filament so that the myosin motor can experience tension. Through this cross-talk, myosin II and cortexillin I define a cellular-scale mechanosensor that monitors and corrects shape defects, ensuring symmetrical cell division.

Understanding the Cooperative Interaction between Myosin II and Actin Cross-Linkers Mediated by Actin Filaments during Mechanosensation

Myosin II is a central mechanoenzyme in a wide range of cellular morphogenic processes. Its cellular localization is dependent not only on signal transduction pathways, but also on mechanical stress. We suggest that this stress-dependent distribution is the result of both the force-dependent binding to actin filaments and cooperative interactions between bound myosin heads. By assuming that the binding of myosin heads induces and/or stabilizes local conformational changes in the actin filaments that enhances myosin II binding locally, we successfully simulate the cooperative binding of myosin to actin observed experimentally. In addition, we can interpret the cooperative interactions between myosin and actin cross-linking proteins observed in cellular mechanosensation, provided that a similar mechanism operates among different proteins. Finally, we present a model that couples cooperative interactions to the assembly dynamics of myosin bipolar thick filaments and that accounts for the transient behaviors of the myosin II accumulation during mechanosensation. This mechanism is likely to be general for a range of myosin II-dependent cellular mechanosensory processes.

Competition between human cells by entosis

Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing (“winner”) and engulfed (“loser”) cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors.

A mechanosensory system governs myosin II accumulation in dividing cells.

A mechanosensory system is characterized that fine-tunes the level of myosin II at the cleavage furrow. This mechanosensory system consists of the mechanosensory module composed of myosin II and cortexillin I and a mechanotransduction loop that includes IQGAP2, kinesin-6, and INCENP.

Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation

Abnormalities in the huntingtin protein (Htt) are associated with Huntingtons disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt(-) cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt(-) cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.

Pharmacological activation of myosin II paralogs to correct cell mechanics defects

Significance Despite the integral role of cell mechanics, efforts to target mechanics for drug development have lagged. Here, we present an approach to identifying small molecules capable of modulating mechanics. We characterize 4-hydroxyacetophenone (4-HAP), isolated as a breakdown product of a hit from our pilot screen of over 22,000 compounds. We show that 4-HAP specifically alters the localization of the mechanoenzyme myosin II, increasing the stiffness of cells. The effect of 4-HAP on myosin II, whose specificity we have defined, occurs across phylogeny. In particular, we have demonstrated 4-HAP’s ability to convert the mechanical profile of metastasis-derived pancreatic cancer cells toward a normal WT-like state. Invasion and migration of these cells, which are hallmarks of the invasive capacity of malignant lesions, are decreased by 4-HAP. Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.

Yes-Associated Protein impacts adherens junction assembly through regulating actin cytoskeleton organization

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

Mimicking the mechanical properties of the cell cortex by the self-assembly of an actin cortex in vesicles.

The composite of the actin cytoskeleton and plasma membrane plays important roles in many biological events. Here, we employed the emulsion method to synthesize artificial cells with biomimetic actin cortex in vesicles and characterized their mechanical properties. We demonstrated that the emulsion method provides the flexibility to adjust the lipid composition and protein concentrations in artificial cells to achieve the desired size distribution, internal microstructure, and mechanical properties. Moreover, comparison of the cortical elasticity measured for reconstituted artificial cells to that of real cells, including those manipulated using genetic depletion and pharmacological inhibition, strongly supports that actin cytoskeletal proteins are dominant over lipid molecules in cortical mechanics. Our study indicates that the assembly of biological systems in artificial cells with purified cellular components provides a powerful way to answer biological questions.

Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation

Genetic interaction analysis is used to identify new cytokinesis proteins involved in myosin II cleavage furrow accumulation and to demonstrate how different pathways collaborate to drive myosin II to the cleavage furrow. One of these proteins, RMD1, is required for myosin II cleavage furrow localization and acts in parallel with mechanical stress.