PDGF-B secreted from skeletal muscle enhances myoblast proliferation and myotube maturation via activation of the PDGFR signaling cascade.

Myokines, secreted factors from skeletal muscle, act locally on muscle cells or satellite cells, which is important in regulating muscle mass and function. Here, we found platelet-derived growth factor subunit B (PDGF-B) is constitutively secreted from muscle cells without muscle contraction. Furthermore, PDGF-B secretion increased with myoblast to myotube differentiation. To examine the role of PDGF-B as a paracrine or autocrine myokine, myoblasts or myotubes were treated with PDGF-B. As a result, myoblast proliferation was significantly enhanced via several signaling pathways. Intriguingly, myotubes treated with PDGF-B showed enhanced maturation as indicated by their increased myotube diameter, myosin heavy chain expression, and strengthened contractile force. These findings suggest that PDGF-B is constitutively secreted by myokines to enhance myoblast proliferation and myotube maturation, which may contribute to skeletal muscle regeneration.

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

Polarized light retardation analysis allows for the evaluation of tension in individual stress fibers.

The tension in the stress fibers (SFs) of cells plays a pivotal role in determining biological processes such as cell migration, morphological formation, and protein synthesis. Our previous research developed a method to evaluate the cellular contraction force generated in SFs based on photoelasticity-associated retardation of polarized light; however, we employed live cells, which could have caused an increase in retardation and not contraction force. Therefore, the present study aimed to confirm that polarized light retardation increases inherently due to contraction, regardless of cell activity. We also explored the reason why retardation increased with SF contractions. We used SFs physically isolated from vascular smooth muscle cells to stop cell activity. The retardation of SFs was measured after ATP administration, responsible for contracting SFs. The SFs were imaged under optical and electron microscopes to measure SF length, width, and retardation. The retardation of isolated SFs after ATP administration was significantly higher than before. Thus, we confirmed that retardation increased with elevated tension in individual SFs. Furthermore, the SF diameter decreased while the SF length remained almost constant. Thus, we conclude that a contraction force-driven increase in the density of SFs is the main factor for the rise in polarized light retardation.

Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells.

Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.

Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers.

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.

Comprehensive analysis on the whole Rho-GAP family reveals that ARHGAP4 suppresses EMT in epithelial cells under negative regulation by Septin9.

Epithelial to mesenchymal transition (EMT) is a fundamental biological process that occurs during development and tumorigenesis. The Rho family of GTPases (Rho-family) is a well-characterized regulator of actin cytoskeleton that gives rise to EMT-associated cell activities. Meanwhile, there are in total at least 66 different Rho-GTPase-activating proteins (Rho-GAPs), which, as an upstream regulator, inactivate specific members of the Rho-family in a cell context-dependent manner. However, molecular roles of individual Rho-GAPs are poorly understood, particularly regarding their involvements in EMT. Here, based on comprehensive screening on the whole Rho-GAP family, we identified specific Rho-GAPs that are responsible for the maintenance of epithelial cell phenotypes, suppressing EMT in human mammary epithelial cells. Specifically, we revealed that at least two Rho-GAPs, that is, ARHGAP4 and SH3BP1, critically regulate the cell morphology. Among them, we focused on ARHGAP4 and demonstrated with multidisciplinary approaches that this specific Rho-GAP regulates epithelial/mesenchymal-selective marker expression, cell proliferation, migration, 3D morphogenesis, and focal adhesion/stress fiber-driven physical force generation in a manner reminiscent of the EMT process. Furthermore, we identified Septin9 with proteomic analyses as a negative regulator of ARHGAP4, which promotes the occurrence of EMT by activation of the FAK/Src signaling pathway. These findings shed light on the novel Rho-GAP-associated pathway in the EMT process under development and tumorigenesis.

Image based cellular contractile force evaluation with small-world network inspired CNN: SW-UNet.

We propose an image based cellular contractile force evaluation method using a machine learning technique. We use a special substrate that exhibits wrinkles when cells grab the substrate and contract, and the wrinkles can be used to visualize the force magnitude and direction. In order to extract wrinkles from the microscope images, we develop a new CNN (convolutional neural network) architecture SW-UNet (small-world U-Net), which is a CNN that reflects the concept of the small-world network. The SW-UNet shows better performance in wrinkle segmentation task compared to other methods: the error (Euclidean distance) of SW-UNet is 4.9 times smaller than the 2D-FFT (fast Fourier transform) based segmentation approach, and is 2.9 times smaller than U-Net. As a demonstration, here we compare the contractile force of U2OS (human osteosarcoma) cells and show that cells with a mutation in the KRAS oncogene show larger force compared to wild-type cells. Our new machine learning based algorithm provides us an efficient, automated and accurate method to evaluate the cell contractile force.

Multi-well plate cell contraction assay detects negatively correlated cellular responses to pharmacological inhibitors in contractility and migration.

To enable large-scale screening of signaling molecules and drugs that regulate cellular contractility-associated mechanotransduction, we previously modified, particularly in terms of the capability of efficiently collecting big data, conventional methodologies using wrinkled substrates to determine the cellular contractility. Here, we present a new system to perform the wrinkle-based cell force assay in a multi-well plate format conformed to standardized geometric configurations and compatible with available technologies such as automated plate readers. With this highly improved throughput in terms of hardware as well as software using a deep learning-based technology, we evaluated the effect of treating cells with various types of pharmacological inhibitors on the cellular contractility. We found opposite responses of cells to the inhibitors between the contractility and collective migration activities. While similar inverse relationships between the contractility and migration have been reported in separate studies, our results here with the high-throughput screening system more broadly generalized these observations.

Keratin-binding ability of the N-terminal Solo domain of Solo is critical for its function in cellular mechanotransduction.

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.

Spatially selective myosin regulatory light chain regulation is absent in dedifferentiated vascular smooth muscle cells but is partially induced by fibronectin and Klf4.

The phosphorylation state of myosin regulatory light chain (MRLC) is central to the regulation of contractility that impacts cellular homeostasis and fate decisions. Rho-kinase (ROCK) and myosin light chain kinase (MLCK) are major kinases for MRLC documented to selectively regulate MRLC in a subcellular position-specific manner; specifically, MLCK in some nonmuscle cell types works in the cell periphery to promote migration, while ROCK does so at the central region to sustain contractility. However, it remains unclear whether or not the spatially selective regulation of the MRLC kinases is universally present in other cell types, including dedifferentiated vascular smooth muscle cells (SMCs). Here, we demonstrate the absence of the spatial regulation in dedifferentiated SMCs using both cell lines and primary cells. Thus, our work is distinct from previous reports on cells with migratory potential. We also observed that the spatial regulation is partly induced upon fibronectin stimulation and Krüppel-like factor 4 overexpression. To find clues to the mechanism, we reveal how the phosphorylation state of MRLC is determined within dedifferentiated A7r5 SMCs under the enzymatic competition among three major regulators ROCK, MLCK, and MRLC phosphatase (MLCP). We show that ROCK, but not MLCK, predominantly regulates the MRLC phosphorylation in a manner distinct from previous in vitro-based and in silico-based reports. In this ROCK-dominating cellular system, the contractility at physiological conditions was regulated at the level of MRLC diphosphorylation, because its monophosphorylation is already saturated. Thus, the present study provides insights into the molecular basis underlying the absence of spatial MRLC regulation in dedifferentiated SMCs.

Vinexin family (SORBS) proteins play different roles in stiffness-sensing and contractile force generation.

Vinexin, c-Cbl associated protein (CAP) and Arg-binding protein 2 (ArgBP2) constitute an adaptor protein family called the vinexin (SORBS) family that is targeted to focal adhesions (FAs). Although numerous studies have focused on each of the SORBS proteins and partially elucidated their involvement in mechanotransduction, a comparative analysis of their function has not been well addressed. Here, we established mouse embryonic fibroblasts that individually expressed SORBS proteins and analysed their functions in an identical cell context. Both vinexin-α and CAP co-localized with vinculin at FAs and promoted the appearance of vinculin-rich FAs, whereas ArgBP2 co-localized with α-actinin at the proximal end of FAs and punctate structures on actin stress fibers (SFs), and induced paxillin-rich FAs. Furthermore, both vinexin-α and CAP contributed to extracellular matrix stiffness-dependent vinculin behaviors, while ArgBP2 stabilized α-actinin on SFs and enhanced intracellular contractile forces. These results demonstrate the differential roles of SORBS proteins in mechanotransduction.

Transgelin-1 (SM22α) interacts with actin stress fibers and podosomes in smooth muscle cells without using its actin binding site.

Transgelin-1 (SM22α) has been recognized as a smooth muscle marker and a tumor suppressor, but many details of the working mechanisms remain unclear. Transgelin-1 belongs to the calponin family of actin-binding proteins with an N-terminal calponin homology domain (CH-domain) and a C-terminal calponin-like module (CLIK23). Here, we demonstrate that transgelin-1 interacts with actin stress fibers and podosomes in smooth muscle cells via its type-3 CH-domain, while CLIK23 is dispensable for the binding to the actin structures. We further suggest that the EF-hand motif in transgelin-1 contributes to proper folding of the CH-domain and in turn to the interaction with the actin structures. These results are in contrast to the ones reported in in vitro studies that demonstrated CLIK23 was necessary for the transgelin-1-actin binding, while the CH-domain was not. Besides, within cells, transgelin-1 phosphorylation at Ser181 in CLIK23 did not affect its colocalization with the actin structures, while the same phosphorylation was reported in in vitro studies to negatively regulate actin binding. Thus, our results suggest the molecular basis of intracellular interaction between transgelin-1 and actin, distinct from that in vitro. The actin binding capability intrinsic to CLIK23 may not appear within cells probably because of the weaker competition for actin binding compared to other actin binding molecules.

New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration.

Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale.

Cellular force assay detects altered contractility caused by a nephritis-associated mutation in nonmuscle myosin IIA.

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II (NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney (HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease.

Contact guidance of smooth muscle cells is associated with tension-mediated adhesion maturation.

Contact guidance is a cellular phenomenon observed during wound healing and developmental patterning, in which adherent cells align in the same direction due to physical cues. Despite numerous studies, the molecular mechanism underlying the consistent cell orientation is poorly understood. Here we fabricated microgrooves with a pitch of submicrons to study contact guidance of smooth muscle cells. We show that both integrin-based cell-substrate adhesions and cellular tension are necessary to achieve contact guidance along microgrooves. We further show through analyses on paxillin that cell-substrate adhesions are more prone to become mature when they run along microgrooves than align at an angle to the direction of microgrooves. Because cellular tension promotes the maturation of cell-substrate adhesions, we propose that the adhesions aligning across microgrooves are not physically efficient for bearing cellular tension compared to those aligning along microgrooves. Thus, the proposed model describes a mechanism of contact guidance that cells would finally align preferentially along microgrooves because cellular tensions are more easily borne within the direction, and the direction of resulting mature adhesions determines the direction of the whole cells.

Aligning cells in arbitrary directions on a membrane sheet using locally formed microwrinkles.

Sheets of cells can be used for tissue regenerative medicine. Cell alignment within the sheet is now a key factor in the next generation of this technology. Anisotropic cell sheets without random cell orientations have been conventionally produced with photolithographically, microfabricated substrates using special facilities and equipment. Here we demonstrate a more accessible approach to the fabrication of anisotropic substrates. We locally deformed part of an elastic membrane and simultaneously oxidized the surface to create microwrinkles as well as to enable adhesion to the extracellular matrix. The approach with the local loading made it possible to orient cells in controlled directions within a single membrane sheet depending on the strains determined by the controllable deformation. This technique potentially enables a versatile design of microwrinkles for target-compatible cell alignments.

Development of motorized plasma lithography for cell patterning.

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.

Intracellular Macromolecular Crowding within Individual Stress Fibers Analyzed by Fluorescence Correlation Spectroscopy.

Introduction: The diffusion of cell components such as proteins is crucial to the function of all living cells. The abundance of macromolecules in cells is likely to cause a state of macromolecular crowding, but its effects on the extent of diffusion remain poorly understood. Methods: Here we investigate the diffusion rate in three distinct locations in mesenchymal cell types, namely the open cytoplasm, the stress fibers in the open cytoplasm, and those below the nucleus using three kinds of biologically inert green fluorescent proteins (GFPs), namely a monomer, dimer, and trimer GFP. Fluorescence correlation spectroscopy (FCS) was used to determine the diffusion coefficients. Results: We show that diffusion tends to be lowered on average in stress fibers and is significantly lower in those located below the nucleus. Our data suggest that the diffusive properties of GFPs, and potentially other molecules as well, are hindered by macromolecular crowding. However, although the size dependence on protein diffusion was also studied for monomer, dimer, and trimer GFPs, there was no significant difference in the diffusion rates among the GFPs of these sizes. These results could be attributed to the lack of significant change in protein size among the selected GFP multimers. Conclusion: The data presented here would provide a basis for better understanding of the complex protein diffusion in the nonuniform cytoplasm, shedding light on cellular responses to mechanical stress, their local mechanical properties, and reduced turnover in senescent cells.

High extensibility of stress fibers revealed by in vitro micromanipulation with fluorescence imaging.

Stress fibers (SFs), subcellular bundles of actin and myosin filaments, are physically connected at their ends to cell adhesions. The intracellular force transmitted via SFs plays an essential role in cell adhesion regulation and downstream signaling. However, biophysical properties intrinsic to individual SFs remain poorly understood partly because SFs are surrounded by other cytoplasmic components that restrict the deformation of the embedded materials. To characterize their inherent properties independent of other structural components, we isolated SFs from vascular smooth muscle cells and mechanically stretched them by in vitro manipulation while visualizing strain with fluorescent quantum dots attached along their length. SFs were elongated along their entire length, with the length being approximately 4-fold of the stress-free length. This surprisingly high extensibility was beyond that explained by the tandem connection of actin filaments and myosin II bipolar filaments present in SFs, thus suggesting the involvement of other structural components in their passive biophysical properties.

Simultaneous contraction and buckling of stress fibers in individual cells.

It has been proposed that buckling of actin stress fibers (SFs) may be associated with their disassembly. However, much of the detail remains unknown partly because the use of an elastic membrane sheet, conventionally necessary for inducing SF buckling with a mechanical compression to adherent cells, may limit high quality and quick imaging of the dynamic cellular events. Here, we present an alternate approach to induce buckling behavior of SFs on a readily observable glass plate. Actin SFs were extracted from cells, and constituent myosin II (MII) molecules were partially photo-inactivated in contractility. An addition of Mg-ATP allowed actin-myosin cross-bridge cycling and resultant contraction of only thick SFs that still contained active MII in the large volume. Meanwhile, thin SFs with virtually no active motor protein in the small volume had no choice but to buckle with the shortening movement of nearby thick SFs functioning as a compression-inducing element. This novel technique, thus allowing for selective inductions of contraction and buckling of SFs and measurements of the cellular prestress, may be applicable to not only investigations on their disassembly mechanisms but also to measurements of the relative thickness of individual SFs in each cell.