Actomyosin Stress Fibers Research Articles

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo, and explored how spermathecal tissue responds to alterations in myosin activity. In animals expressing GFP labeled actin or apical membrane complexes, we severed these structures using femtosecond laser ablation and quantified retractions. RNA interference was used to deplete key contractility regulators. We show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. Our results suggest that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. Additionally, increased tension on the outer basal surface may compress the apical side, leading to lower tensions apically. The three dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

Arpin deficiency increases actomyosin contractility and vascular permeability

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

Arpin deficiency increases actomyosin contractility and vascular permeability

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

A chemo-mechanical model for growth and mechanosensing of focal adhesion

Focal adhesion (FA), the complex molecular assembly across the lipid membrane, serves as a hub for physical and chemical information exchange between cells and their microenvironment. Interestingly, studies have shown that FAs can grow along the direction of contractile forces generated by actomyosin stress fibers and achieve larger sizes on stiffer substrates. In addition, the cellular traction transmitted to the substrate was observed to reach the maximum near the FA center. However, the biomechanical mechanisms behind these intriguing findings remain unclear. To answer this important question, here we first developed a one-dimensional (1D) chemo-mechanical model of FA where key features like adhesion plaque deformation, active contraction by stress fibers, force-dependent association/dissociation of integrin bonds connecting two surfaces, and substrate compliance have all been considered. Within this formulation, we showed that the rigidity-sensing capability of FAs originates from the deformability of stress fibers while the force-dependent breakage of integrin bonds leads to the appearance of the traction peak at the FA center. Furthermore, by extending the model into three-dimensional as well as incorporating assembly/dis-assembly kinetics of adhesion proteins, we also demonstrated how anisotropic stress/strain field within the adhesion plaque will be induced by the presence of contractile forces which leads to the directional growth of the FA.

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

Molecular profiling of sponge deflation reveals an ancient relaxant-inflammatory response

A hallmark of animals is the coordination of whole-body movement. Neurons and muscles are central to this, yet coordinated movements also exist in sponges that lack these cell types. Sponges are sessile animals with a complex canal system for filter-feeding. They undergo whole-body movements resembling "contractions" that lead to canal closure and water expulsion. Here, we combine live 3D optical coherence microscopy, pharmacology, and functional proteomics to elucidate the sequence and detail of shape changes, the tissues and molecular physiology involved, and the control of these movements. Morphometric analysis and targeted perturbation suggest that the movement is driven by the relaxation of actomyosin stress fibers in epithelial canal cells, which leads to whole-body deflation via collapse of the incurrent and expansion of the excurrent canal system. Thermal proteome profiling and quantitative phosphoproteomics confirm the control of cellular relaxation by an Akt/NO/PKG/PKA pathway. Agitation-induced deflation leads to differential phosphorylation of proteins forming epithelial cell junctions, implying their mechanosensitive role. Unexpectedly, untargeted metabolomics detect a concomitant decrease in antioxidant molecules during deflation, reflecting an increase in reactive oxygen species. Together with the secretion of proteinases, cytokines, and granulin, this indicates an inflammation-like state of the deflating sponge reminiscent of vascular endothelial cells experiencing oscillatory shear stress. These results suggest the conservation of an ancient relaxant-inflammatory response of perturbed fluid-carrying systems in animals and offer a possible mechanism for whole-body coordination through diffusible paracrine signals and mechanotransduction.

BORG family proteins in physiology and human disease.

The binder of rho GTPases (BORG)/Cdc42 effector proteins (Cdc42EP) family is composed of five Rho GTPase binding proteins whose functions and mechanism of actions are of emerging interest. Here, we review recent findings pertaining to the family as a whole and consider how these change our understanding of cellular organization. Recent studies have implicated BORGs in both fundamental physiology and in human diseases, mainly cancers. An emerging pattern suggests that BORG family members cancer-promoting properties are related to their ability to regulate the cytoskeleton, with many impacting the organization of acto-myosin stress fibers. This is consistent with the broader literature indicating that BORG family members are regulators of both the septin and actin cytoskeleton networks. The exact mechanism through which BORGs modify the cytoskeleton is not clear, but we consider here a few data-supported and speculative possibilities. Finally, we delve into how the Rho GTPase Cdc42 modifies BORG function in cells. This remains open-ended as Cdc42's effects on BORGs appear cell type- and cell state-dependent. Collectively, these data point to the importance of the BORG family and suggest broader themes in their function and regulation.

Three-dimensional analysis of hydrogel-imbedded aortic valve interstitial cell shape and its relation to contractile behavior.

Cell-shape is a conglomerate of mechanical, chemical, and biological mechanisms that reflects the cell biophysical state. In a specific application, we consider aortic valve interstitial cells (AVICs), which maintain the structure and function of aortic heart valve leaflets. Actomyosin stress fibers help determine AVIC shape and facilitate processes such as adhesion, contraction, and mechanosensing. However, detailed 3D assessment of stress fiber architecture and function is currently impractical. Herein, we assessed AVIC shape and contractile behaviors using hydrogel-based 3D traction force microscopy to intuit the orientation and behavior of AVIC stress fibers. We utilized spherical harmonics (SPHARM) to quantify AVIC geometries through three days of incubation, which demonstrated a shift from a spherical shape to forming substantial protrusions. Furthermore, we assessed changes in post-three day AVIC shape and contractile function within two testing regimes: (1) normal contractile level to relaxation (cytochalasin D), and (2) normal contractile level to hyper-contraction (endothelin-1). In both scenarios, AVICs underwent isovolumic shape changes and produced complex displacement fields within the hydrogel. AVICs were more elongated when relaxed and more spherical in hyper-contraction. Locally, AVIC protrusions contracted along their long axis and expanded in their circumferential direction, indicating predominately axially aligned stress fibers. Furthermore, the magnitude of protrusion displacements was correlated with protrusion length and approached a consistent displacement plateau at a similar critical length across all AVICs. This implied that stress fiber behavior is conserved, despite great variations in AVIC shapes. We anticipate our findings will bolster future investigations into AVIC stress fiber architecture and function. STATEMENT OF SIGNIFICANCE: Within the aortic valve there exists a population of aortic valve interstitial cells, which orchestrate the turnover, secretion, and remodeling of its extracellular matrix, maintaining tissue integrity and ultimately sustaining the proper mechanical function. Alterations in these processes are thought to underlie diseases of the aortic valve, which affect hundreds of thousands domestically and world-wide. Yet, to date, there are no non-surgical treatments for aortic heart valve disease, in part due to our limited understanding of the underlying disease processes. In the present study, we built upon our previous study to include a full 3D analysis of aortic valve interstitial cell shapes at differing contractile levels. The resulting detailed shape and deformation analysis provided insight into the underlying stress-fiber structures and mechanical behaviors.

Actin crosslinking by α-actinin averts viscous dissipation of myosin force transmission in stress fibers

Contractile force generated in actomyosin stress fibers (SFs) is transmitted along SFs to the extracellular matrix (ECM), which contributes to cell migration and sensing of ECM rigidity. In this study, we show that efficient force transmission along SFs relies on actin crosslinking by α-actinin. Upon reduction of α-actinin-mediated crosslinks, the myosin II activity induced flows of actin filaments and myosin II along SFs, leading to a decrease in traction force exertion to ECM. The fluidized SFs maintained their cable integrity probably through enhanced actin polymerization throughout SFs. A computational modeling analysis suggested that lowering the density of actin crosslinks caused viscous slippage of actin filaments in SFs and, thereby, dissipated myosin-generated force transmitting along SFs. As a cellular scale outcome, α-actinin depletion attenuated the ECM-rigidity-dependent difference in cell migration speed, which suggested that α-actinin-modulated SF mechanics is involved in the cellular response to ECM rigidity.

Nuclear pore complexes concentrate on Actin/LINC/Lamin nuclear lines in response to mechanical stress in a SUN1 dependent manner

Formation of robust actomyosin stress fibers (SF) in response to cell stretch plays a key role in the transfer of information from the cytoplasm into the nucleus. Actin/LINC/Lamin (ALL) nuclear lines provide mechanical linkage between the actin cytoskeleton and the lamin nucleoskeleton across the nuclear envelope. To understand the establishment of ALL lines, we used live cell imaging of cells exposed to cyclic stretch. We discovered that nuclear pore complexes (NPCs) concentrate along ALL lines that are generated in response to uniaxial cyclic stretch. The ALL-associated NPCs display increased fluorescence intensity of nucleoporins Pom121, TPR and Nup153 relative to nucleoporins that are distal to the ALL lines. Here we test the hypothesis that a LINC complex component of ALL lines, SUN1 is involved in the integration of NPCs with ALL lines. We generated CRISPR SUN1 knockdown and knockout cell lines and show that SUN1 is essential for normal integration of NPCs to ALL lines. Loss or elimination of SUN1 significantly diminishes NPC/ALL line integration, demonstrating a key role for SUN1 in the recruitment or stabilization of NPCs to a discrete subdomain of the nuclear envelope at ALL lines. This work provides new insight into the mechanism by which cells respond to mechanical force through nuclear envelope remodeling.

Extracellular matrix stiffness regulates degradation of MST2 via SCF βTrCP

The Hippo pathway plays central roles in relaying mechanical signals during development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we found that chemical inhibition of proteasomal proteolysis resulted in increased levels of MST2 in human breast epithelial cells. MST2 binds SCFβTrCP E3 ubiquitin ligase and silencing βTrCP resulted in MST2 accumulation. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that βTrCP binds MST2 via a non-canonical degradation motif. Additionally, stiffer extracellular matrix, as well as hyperactivation of integrins resulted in enhanced MST2 degradation mediated by integrin-linked kinase (ILK) and actomyosin stress fibers. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and underscores how alterations in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

Coordination of contractile tension and cell area changes in an epithelial cell monolayer.

During tissue development and repair, cells contract and expand in coordination with their neighbors, giving rise to tissue deformations that occur on length scales far larger than that of a single cell. The biophysical mechanisms by which the contractile forces of each cell cause deformations on multicellular length scales are not fully clear. To investigate this question, we began with the principle of force equilibrium, which dictates a balance of tensile forces between neighboring cells. Based on this principle, we hypothesized that coordinated changes in cell area result from tension transmitted across the cell layer. To test this hypothesis, spatial correlations of both contractile tension and the divergence of cell velocities were measured as readouts of coordinated contractility and collective area changes, respectively. Experiments were designed to alter the spatial correlation of contractile tension using three different methods, including disrupting cell-cell adhesions, modulating the alignment of actomyosin stress fibers between neighboring cells, and changing the size of the cell monolayer. In all experiments, the spatial correlations of both tension and divergence increased or decreased together, in agreement with our hypothesis. To relate our findings to the intracellular mechanism connecting changes in cell area to contractile tension, we disrupted activation of extracellular signal-regulated kinase (ERK), which is known to mediate the intracellular relationship between cell area and contraction. Consistent with prior knowledge, a temporal cross-correlation between cell area and tension revealed that ERK was responsible for a proportional relationship between cell area and contraction. Inhibition of ERK activation reduced the spatial correlations of the divergence of cell velocity but not of tension. Together, our findings suggest that coordination of cell contraction and expansion requires transfer of cell tension over space and ERK-mediated coordination between cell area and contraction in time.

Measuring viscoelastic properties of single stress fibers during 3D confined migration

Actomyosin stress fibers (SFs) allow cells to exert tensile forces on their surrounding microenvironment, which in turn underlies cell structure, mechanics, and migration. While the tensile properties and functional contributions of SFs have been extensively explored in 2D geometries, much remains unknown about how SFs function in 3D, particularly in confined geometries relevant to invasion and metastasis. Here we integrate 3D printing with single-SF femtosecond laser ablation to measure SF viscoelastic properties in well-defined 3D confined geometries.

Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers.

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation.

Anxiety and depression: A matter of stiffness?

Cells react to stress by the universal responses of “fluidization” or “reinforcement” (stiffening) of the cytoplasm, through dramatic re-arrangements of the cytoskeleton. Here it is suggested that, at a supracellular level, the brain exhibits such a fundamental behavior as part of its complex response to stress: it is hypothesized that the soft gel formed by brain cell cytoskeletons and the surrounding extracellular matrix (the “cytoskeletons-matrix system”) undergoes transitions either to sol (fluidization) or stiff gel (reinforcement) as a very fundamental and evolutionarily conserved brain response to stress, alongside more sophisticated neural pathways. Sol state corresponds to increased cell activity (a sort of “fight or flight” response), whereas stiff gel state corresponds to inactivity (an “immobility” strategy).Psychological stress, through simple stress signals such as pH changes, would lead to an initial tissue fluidization in key regions of the brain, followed, if the stress stimuli persist, by reinforcement (slow formation of actomyosin stress fibers and matrix stiffening).It is also hypothesized that the cytoskeletons-matrix system is one of the biological correlates of so-called “background feelings”, i.e conscious feelings built on inner chemical-physical states of the body. Optimal dynamics of the cytoskeletons-matrix system would contribute to a core feeling of well-being, while shifts towards fluidization (activation) or stiffening (inactivation) would contribute to background feelings at the basis of anxiety and stress-induced depression, respectively.It is suggested that the cytoskeletons-matrix system behaves as a “self-organized critical system”, anxiety and depression arising whenever the system is driven too far from the optimal critical point.Finally, some application hints from the proposed ideas are given.

Cofilin is required for polarization of tension in stress fiber networks during migration.

Cell migration is associated with the establishment of defined leading and trailing edges, which in turn requires polarization of contractile forces. While the actomyosin stress fiber (SF) network plays a critical role in enforcing this polarity, precisely how this asymmetry is established remains unclear. Here, we provide evidence for a model in which the actin-severing protein cofilin (specifically cofilin-1) participates in symmetry breakage by removing low-tension actomyosin filaments during transverse arc assembly. Cofilin knockdown (KD) produces a non-polarized SF architecture that cannot be rescued with chemokines or asymmetric matrix patterns. Whereas cofilin KD increases whole-cell prestress, it decreases prestress within single SFs, implying an accumulation of low-tension SFs. This notion is supported by time-lapse imaging, which reveals weakly contractile and incompletely fused transverse arcs. Confocal and super-resolution imaging further associate this failed fusion with the presence of crosslinker-rich, tropomyosin-devoid nodes at the junctions of multiple transverse arc fragments and dorsal SFs. These results support a model in which cofilin facilitates the formation of high-tension transverse arcs, thereby promoting mechanical asymmetry.

Integrins regulate epithelial cell shape by controlling the architecture and mechanical properties of basal actomyosin networks

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.

Biomechanical Response of Lung Epithelial Cells to Iron Oxide and Titanium Dioxide Nanoparticles.

Increasing evidence shows that lungs can be damaged by inhalation of nanoparticles (NPs) at environmental and occupational settings. Recent findings have associated the exposure to iron oxide (Fe2O3) and titanium dioxide (TiO2) – NPs widely used in biomedical and clinical research – with pulmonary oxidative stress and inflammation. Although changes on cellular mechanics could contribute to pulmonary inflammation, there is no information regarding the effects of Fe2O3 and TiO2 on alveolar epithelial cell biomechanics. The aim was to investigate the NPs-induced biomechanical effects in terms of cell stiffness and traction forces exerted by human alveolar epithelial cells. Cell Young’s modulus (E) measured by atomic force microscopy in alveolar epithelial cells significantly decreased after exposure to Fe2O3 and TiO2 (∼28 and ∼25%, respectively) compared to control conditions. Moreover, both NPs induced a similar reduction in the traction forces exerted by the alveolar epithelial cells in comparison to the control conditions. Accordingly, immunofluorescence images revealed a reduction of actomyosin stress fibers in response to the exposure to NPs. However, no inflammatory response was detected. In conclusion, an acute exposure of epithelial pulmonary cells to Fe2O3 and TiO2 NPs, which was mild since it was non-cytotoxic and did not induce inflammation, modified cell biomechanical properties which could be translated into damage of the epithelial barrier integrity, suggesting that mild environmental inhalation of Fe2O3 and TiO2 NPs could not be innocuous.

Multifunctional roles of tropomodulin-3 in regulating actin dynamics.

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.

Rock and MlCK Tune Regional Stress Fiber Mechanics via Preferential Myosin Light Chain Phosphorylation

Non-muscle myosin II (NMMII) is the key force-generating component of actomyosin stress fibers (SFs), which transmit tension throughout the cytoskeleton and to the extracellular matrix. NMMII motor activity is strongly promoted by phosphorylation of its regulatory light chains (RLCs) at Ser19 (p-RLC) and additionally enhanced by further phosphorylation at Thr18 (pp-RLC). The main RLC regulatory kinases are myosin RLC kinase (MLCK) and Rho-associated kinase (ROCK). While prior work suggests that MLCK and ROCK respectively regulate SFs located in the cellular center and periphery, it is unclear how these kinases differ in their effects on RLC phosphorylation state and how each phosphospecies influences the mechanics of central and peripheral SFs. Here we address these questions by combining graded induction of ROCK or MLCK expression with phospho-RLC immuno-analysis and mechanical measurement of single SFs in living cells. Immunoblots and quantitative immunofluorescence imaging reveal that graded MLCK overexpression preferentially produces graded increases in p-RLC, which localizes to peripheral SFs, while graded ROCK overexpression increases pp-RLC, which localizes to central SFs. Single-SF laser ablation studies revealed that MLCK increases the effective viscosity and stored elastic energy in peripheral SFs but not central SFs, while ROCK preferentially increases these parameters in central SFs. Graded variations in kinase activities revealed surprising and previously unappreciated nonlinearities between phosphorylation levels and SF viscoelasticity. To establish causal relationships between RLC phosphospecies and SF mechanics, we overexpressed RLC mono- and di-phosphomimetic mutants. We found that phosphomimetic p-RLC localized to and selectively influenced the mechanics of peripheral SFs while di-phosphomimetic pp-RLC localized to and selectively influenced the mechanics of central SFs. This work supports a model in which MLCK regulates peripheral SF viscoelasticity via p-RLC while ROCK regulates central SF viscoelasticity via pp-MLC.