Cells exhibit various morphological characteristics due to their physiological activities, and changes in cell morphology are inherently accompanied by the assembly and disassembly of the actin cytoskeleton. Stress fibers are a prominent component of the actin-based intracellular structure and are highly involved in numerous physiological processes, e.g., mechanotransduction and maintenance of cell morphology. Although it is widely accepted that variations in cell morphology interact with the distribution and localization of stress fibers, it remains unclear if there are underlying geometric principles between the cell morphology and actin cytoskeleton. Here, we present a machine learning system that uses the diffusion model to convert the cell shape to the distribution and alignment of stress fibers. By training with corresponding cell shape and stress fibers datasets, our system learns the conversion to generate the stress fiber images from its corresponding cell shape. The predicted stress fiber distribution agrees well with the experimental data. With this conversion relation, our system allows for performing virtual experiments that provide a visual map showing the probability of stress fiber distribution from the virtual cell shape. Our system potentially provides a powerful approach to seek further hidden geometric principles regarding how the configuration of subcellular structures is determined by the boundary of the cell structure; for example, we found that the stress fibers of cells with small aspect ratios tend to localize at the cell edge while cells with large aspect ratios have homogenous distributions.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Cells dynamically remodel their internal structures by modulating the arrangement of actin filaments (AFs). In this process, individual AFs exhibit stochastic behavior without knowing the macroscopic higher-order structures they are meant to create or disintegrate, but the mechanism allowing for such stochastic process-driven remodeling of subcellular structures remains incompletely understood. Here we employ percolation theory to explore how AFs interacting only with neighboring ones without recognizing the overall configuration can nonetheless create a substantial structure referred to as stress fibers (SFs) at particular locations. We determined the interaction probabilities of AFs undergoing cellular tensional homeostasis, a fundamental property maintaining intracellular tension. We showed that the duration required for the creation of SFs is shortened by the increased amount of preexisting actin meshwork, while the disintegration occurs independently of the presence of actin meshwork, suggesting that the coexistence of tension-bearing and non-bearing elements allows cells to promptly transition to new states in accordance with transient environmental changes. The origin of this asymmetry between creation and disintegration, consistently observed in actual cells, is elucidated through a minimal model analysis by examining the intrinsic nature of mechano-signal transmission. Specifically, unlike the symmetric case involving biochemical communication, physical communication to sense environmental changes is facilitated via AFs under tension, while other free AFs dissociated from tension-bearing structures exhibit stochastic behavior. Thus, both the numerical and minimal models demonstrate the essence of intracellular percolation, in which macroscopic asymmetry observed at the cellular level emerges not from microscopic asymmetry in the interaction probabilities of individual molecules, but rather only as a consequence of the manner of the mechano-signal transmission. These results provide novel insights into the role of the mutual interplay between distinct subcellular structures with and without tension-bearing capability. Insight: Cells continuously remodel their internal elements or structural proteins in response to environmental changes. Despite the stochastic behavior of individual structural proteins, which lack awareness of the larger subcellular structures they are meant to create or disintegrate, this self-assembly process somehow occurs to enable adaptation to the environment. Here we demonstrated through percolation simulations and minimal model analyses that there is an asymmetry in the response between the creation and disintegration of subcellular structures, which can aid environmental adaptation. This asymmetry inherently arises from the nature of mechano-signal transmission through structural proteins, namely tension-mediated information exchange within cells, despite the stochastic behavior of individual proteins lacking asymmetric characters in themselves.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

In the development of chronic liver disease, the hepatic stellate cell (HSC) plays a pivotal role in increasing intrahepatic vascular resistance (IHVR) and inducing portal hypertension (PH) in cirrhosis. Our research demonstrated that HSC contraction, prompted by angiotensin II (Ang II), significantly contributed to the elevation of type I collagen (COL1A1) expression. This increase was intimately associated with enhanced cell tension and YAP nuclear translocation, mediated through α-smooth muscle actin (α-SMA) expression, microfilaments (MF) polymerization, and stress fibers (SF) assembly. Further investigation revealed that the Rho/ROCK signaling pathway regulated MF polymerization and SF assembly by facilitating the phosphorylation of cofilin and MLC, while Ca2+ chiefly governed SF assembly via MLC. Inhibiting α-SMA-MF-SF assembly changed Ang II-induced cell contraction, YAP nuclear translocation, and COL1A1 expression, findings corroborated in cirrhotic mice models. Overall, our study offers insights into mitigating IHVR and PH through cell mechanics, heralding potential breakthroughs.

Epithelial tissue forms and maintains a critical barrier function in the body. A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. Culturing Madin-Darby canine kidney (MDCK) cells at the liquid-liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid-liquid interfaces, cells cultured on a fibronectin-coated liquid-liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid-liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, FZ = 0.73). In Y27632-exposed cells on the liquid-liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as HLN, was 0.93 ± 0.01, indicated even cell distribution in the culture vessel at t = 72 h. The behavior of epithelial cells on liquid-liquid interfaces contributes to the promotion of their uniform maturation.