LUZP1 regulates the maturation of contractile actomyosin bundles.

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.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension.

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.

Na-caprate-induced increase in MDCK II epithelial cell leak pathway permeability and opening number is associated with disruption of basal F-actin organization.

Confluent populations of the epithelial cell line, MDCK II, develop circumferential tight junctions joining adjacent cells to create a barrier to the paracellular movement of solutes and water. Treatment of MDCK II cell populations from the apical surface with 1 mM Na-caprate increased permeability to macromolecules (Leak Pathway) without increasing monolayer disruption or cell death. Graphical analysis of the apparent permeability versus solute Stokes radius for a size range of fluorescein-dextran species indicates apical 1 mM Na-caprate enhances Leak Pathway permeability by increasing the number of Leak Pathway openings without significantly affecting opening size. Na-caprate treatment did not alter the content of any tight junction protein examined. Treatment of MDCK II cell populations with apical 1 mM Na-caprate disrupted basal F-actin stress fibers and decreased the tortuosity of the tight junctions. Treatment of MDCK II cell populations with blebbistatin, a myosin ATPase inhibitor, alone had little effect on Leak Pathway permeability but synergistically increased Leak Pathway permeability when added with 1 mM Na-caprate. Na-caprate exhibited a similar ability to increase Leak Pathway permeability in wild-type MDCK II cell monolayers and ZO-1 knockdown MDCK II cell monolayers but an enhanced ability to increase Leak Pathway permeability in monolayers of TOCA-1 knockout MDCK II cells. These results demonstrate that Na-caprate increases MDCK II cell population Leak Pathway permeability by increasing the number of Leak Pathway openings. This action is likely mediated by alterations in F-actin organization, primarily involving disruption of basal F-actin stress fibers.NEW & NOTEWORTHY This study determines the underlying change in the openings in the epithelial tight junction permeability barrier structure that leads to a change in the paracellular permeability to macromolecules (the Leak Pathway) and connects this to disruption of specific F-actin structures within the cells. It provides important and novel insights into how tight junction permeability to macromolecules is modulated by specific changes to cellular and tight junction composition/organization.

A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments.

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

Mechanical stretch facilitates tenomodulin expression to induce tenocyte migration via MAPK signaling pathway.

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration.

GCKIII (Germinal Center Kinase III) Kinases STK24 and STK25 (Serine/Threonine Kinase 24 and 25) Inhibit Cavernoma Development.

BACKGROUND: Cavernous cerebral malformations can arise because of mutations in the CCM1, CCM2, or CCM3 genes, and lack of Cdc42 has also been reported to induce these malformations in mice. However, the role of the CCM3 (cerebral cavernous malformation 3)-associated kinases in cavernoma development is not known, and we, therefore, have investigated their role in the process. METHODS: We used a combination of an in vivo approach, using mice genetically modified to be deficient in the CCM3-associated kinases STK24 and STK25 (serine/threonine kinases 24 and 25), and the in vitro model of human endothelial cells in which expression of STK24 and STK25 was inhibited by RNA interference. RESULTS: Mice deficient for both Stk24 and Stk25, but not for either of them individually, developed aggressive vascular lesions with the characteristics of cavernomas at an early age. Stk25 deficiency also gave rise to vascular anomalies in the context of Stk24 heterozygosity. Human endothelial cells deficient for both kinases phenocopied several of the consequences of CCM3 loss, and single STK25 deficiency also induced KLF2 expression, Golgi dispersion, altered distribution of ß-catenin, and appearance of stress fibers. CONCLUSIONS: The CCM3-associated kinases STK24 and STK25 play a major role in the inhibition of cavernoma development.

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.

Compressive forces driven by lateral actin fibers are a key to the nuclear deformation under uniaxial cell-substrate stretching.

Cells sense the direction of mechanical stimuli including substrate stretching and show morphological and functional responses. The nuclear deformation with respect to the direction of mechanical stimuli is thought of as a vital factor in mechanosensitive intracellular signaling and gene transcription, but the detailed relationship between the direction of stimuli and nuclear deformation behavior is not fully solved yet. Here, we assessed the role of actin cytoskeletons in nuclear deformation caused by cell substrate stretching with different directions. Cells on a PDMS stretching chamber were subjected to a step-strain and changes of long- and short-axes of nucleus before and after stretching were evaluated in terms of nuclear orientation against the direction of stretching. Nuclei oriented parallel to the stretching direction showed elongation and shrinkage in the long and short axes, respectively, and vice versa. However, calculation of the aspect ratio (ratio of long- and short-axes) changes revealed orientation-depend nuclear deformation: The nucleus oriented parallel to the stretching direction showed a greater aspect ratio change than it aligned in the perpendicular direction of the stretching. A decrease in actin cytoskeletal tension significantly changed the nuclear deformation only in the short axis direction, thereby abolishing the orientation-depend deformation of the nucleus. These results suggest that lateral compressive forces exerted by the actin cytoskeleton is a key factor of orientation-depend deformation in short axis of the nucleus under the cell-substrate stretching condition, and may be crucial for mechano-sensing and responses to the cell-substrate stretching direction.

Spatial organization and crosstalk of vimentin and actin stress fibers regulate the osteogenic differentiation of human adipose-derived stem cells.

Human adipose-derived stem cells (hASCs) are ideal seed cells for tissue engineering due to their multidirectional differentiation potential. Microfilaments, microtubules, and intermediate filaments are responsible for supporting the intracellular space. Vimentin, a type III intermediate filament protein that is specifically expressed in cells of mesenchymal origin, can function as a scaffold and endow cells with tension and shear stress resistance. Actin stress fibers (ASF) act as an important physical device in stress signal transduction, providing stiffness for cells, and promoting osteogenesis. Through direct physical contact, cross-linkers, and spatial interactions, vimentin and actin networks exist as intersecting entities. Spatial interactions occur in the overlapping area of cytoskeleton subsystems, which could affect cell morphology, cell mechanics, and cell fate. However, how does the spatial organization between the cytoskeletal subsystems changed during osteogenesis, especially between vimentin and ASF, is still not understood, and its mechanism effect on cell fate remains unclear. In our study, WB experiment was used to detect the expression changes in Vimentin, ASF, and other proteins. Cells were reconstructed by three-dimensional scanning with fluorescence microscope, and the spatial thickness of vimentin and ASF cytoskeletons and the thickness of the overlapping area between them were calculated, respectively, so as to observe the spatial reorganization of vimentin and ASF in cells. Cytochalasin D (an inhibitor of actin polymerization) and vimentin upregulated/downregulated cells were used to verify the change in the spatial organization between vimentin and ASF and its influence on osteogenesis. Then, heat shock protein 27 (HSP27) was downregulated to illuminate the regulatory mechanisms of spatial organization between vimentin and ASF during osteogenesis. The amounts and the spatial positions of vimentin and actin stress fiber exhibited opposite trends during osteogenesis. Through controlling the anchor sites on the nucleus, intermediate filaments vimentin can reduce the spatial proportion of actin stress fibers, which can be regulated by HSP27. In addition, depolymerization of actin stress fibers lead to lower osteogenic differentiation ability, resulting in osteogenesis and lipogenesis existed simultaneously, that can be resisted by vimentin. Our data indicate that the spatial reorganization of vimentin and actin stress fibers is a key factor in the regulation of the differentiation state of hASCs. And their spatial overlapping area is detrimental to hASCs osteogenesis, providing a new perspective for further exploring the mechanism underlying hASCs osteogenesis.

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.

Actin Stress Fibers Response and Adaptation under Stretch.

One of the many effects of soft tissues under mechanical solicitation in the cellular damage produced by highly localized strain. Here, we study the response of peripheral stress fibers (SFs) to external stretch in mammalian cells, plated onto deformable micropatterned substrates. A local fluorescence analysis reveals that an adaptation response is observed at the vicinity of the focal adhesion sites (FAs) due to its mechanosensor function. The response depends on the type of mechanical stress, from a Maxwell-type material in compression to a complex scenario in extension, where a mechanotransduction and a self-healing process takes place in order to prevent the induced severing of the SF. A model is proposed to take into account the effect of the applied stretch on the mechanics of the SF, from which relevant parameters of the healing process are obtained. In contrast, the repair of the actin bundle occurs at the weak point of the SF and depends on the amount of applied strain. As a result, the SFs display strain-softening features due to the incorporation of new actin material into the bundle. In contrast, the response under compression shows a reorganization with a constant actin material suggesting a gliding process of the SFs by the myosin II motors.

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.

Rhophilin-associated tail protein 1 promotes migration and metastasis in triple negative breast cancer via activation of RhoA.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with high motile and invasive capacity that contributes to metastasis. Understanding the mechanisms for the motility of TNBC might provide novel targetable vulnerabilities of the tumors. Herein, we find that Rhophilin-associated tail protein 1 (ROPN1) is selectively overexpressed in human TNBC cell lines and tissues. Overexpression of ROPN1 promotes, while silencing of ROPN1 inhibits the robust migration, invasion, and in vivo metastasis of TNBC cells. Moreover, we find that ROPN1 activates RhoA signaling via rhophilin-1 (RHPN1), leading to enhanced actin stress fibers formation in TNBC cells. RhoA signaling is demonstrated to be essential for ROPN1-mediated migration and metastasis of TNBC cells. Finally, we find that high levels of ROPN1 are significantly associated distant metastasis and predicted poor prognosis in patients with breast cancer. These findings reveal a novel mechanism for the high motility and metastasis of TNBC cells, suggesting that ROPN1 might be a potential prognostic marker and therapeutic target.

Nanoindentation of mesenchymal stem cells using atomic force microscopy: effect of adhesive cell-substrate structures.

The procedure commonly adopted to characterize cell materials using atomic force microscopy neglects the stress state induced in the cell by the adhesion structures that anchor it to the substrate. In several studies, the cell is considered as made from a single material and no specific information is provided regarding the mechanical properties of subcellular components. Here we present an optimization algorithm to determine separately the material properties of subcellular components of mesenchymal stem cells subjected to nanoindentation measurements. We assess how these properties change if the adhesion structures at the cell-substrate interface are considered or not in the algorithm. In particular, among the adhesion structures, the focal adhesions and the stress fibers were simulated. We found that neglecting the adhesion structures leads to underestimate the cell mechanical properties thus making errors up to 15%. This result leads us to conclude that the action of adhesion structures should be taken into account in nanoindentation measurements especially for cells that include a large number of adhesions to the substrate.

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses.

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2.

Role of boundary conditions in determining cell alignment in response to stretch.

The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.

Stretch-Induced Tenomodulin Expression Promotes Tenocyte Migration via F-Actin and Chromatin Remodeling.

The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd's expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.

Manipulation of Focal Adhesion Signaling by Pathogenic Microbes.

Focal adhesions (FAs) serve as dynamic signaling hubs within the cell. They connect intracellular actin to the extracellular matrix (ECM) and respond to environmental cues. In doing so, these structures facilitate important processes such as cell-ECM adhesion and migration. Pathogenic microbes often modify the host cell actin cytoskeleton in their pursuit of an ideal replicative niche or during invasion to facilitate uptake. As actin-interfacing structures, FA dynamics are also intimately tied to actin cytoskeletal organization. Indeed, exploitation of FAs is another avenue by which pathogenic microbes ensure their uptake, survival and dissemination. This is often achieved through the secretion of effector proteins which target specific protein components within the FA. Molecular mimicry of the leucine-aspartic acid (LD) motif or vinculin-binding domains (VBDs) commonly found within FA proteins is a common microbial strategy. Other effectors may induce post-translational modifications to FA proteins through the regulation of phosphorylation sites or proteolytic cleavage. In this review, we present an overview of the regulatory mechanisms governing host cell FAs, and provide examples of how pathogenic microbes have evolved to co-opt them to their own advantage. Recent technological advances pose exciting opportunities for delving deeper into the mechanistic details by which pathogenic microbes modify FAs.

Rat corneal endothelial cell migration during wound repair on the basement membrane depends more on the PI-3K pathway than the cdc-42 pathway or actin stress fibers.

Following a central transcorneal circular freeze injury, organ-cultured rat corneal endothelial cells surrounding the wound reorganize peripheral actin bands into stress fibers and migrate individually into the wound. To ascertain the significance of this rearrangement relative to morphological changes accompanying migration and wound repair, some tissues were incubated overnight in 4 µM TRITC-conjugated phalloidin to stabilize actin and prevent its reorganization. After a freeze injury to the endothelium tissues were histologically observed at 24 h post-wounding and demonstrated that despite a lack of actin organization, cells responding to the injury appeared morphologically similar to their control counterparts. Tissues cultivated in the presence of either cytochalasin B (CB), soybean agglutinin (SBA), or fluorouracil (FU) and also exhibited actin cytoskeletal disruption. Under these conditions, migration continued despite the absence of detectable stress fibers. For SBA-, CB-, and FU-treated tissues, wound repair did not significantly differ from control preparations although FU-treated tissues showed a slower repair. Electron micrographs confirmed an absence of stress fibers in migrating cells treated with any of these agents. Tissues were also treated with ML 141 and EY294002 to inhibit the cdc-42 and PI-3K pathways, respectively. While cell movement still occurred in the presence of ML 141, migration into the wound was greatly restricted in the presence of EY294002. These results indicate that rat corneal endothelial cell movement in situ does not require actin reorganization into stress fibers, but the functioning of the PI-3K pathway is indispensable for their migration along the basement membrane during wound repair.

An ultrastructural pathologist's views on fibroblasts, modified smooth muscle cells, wound healing, stenosing arteriopathies, Kawasaki disease, Dupuytren's contracture, and the stroma of carcinomas.

It wasn't until 1960 that the dense bodies of the peripheral actin arrays of fibroblasts were finally visualized, i.e., stress fibers (SFs). Mistakenly assumed that its SFs turned the fibroblast into a unique cell situated somewhere in a continuum between it and a smooth muscle cell (SMC), it was descriptively named a "myofibroblast" (MF). Automatically, spindle cells with SFs and/or smooth muscle actin by SMA IHC-staining, became MFs, although endothelial cells, pericytes, modified SMCs (mSMC), and myoepithelial cells all contain SFs. An invisible "intermediate" cell was hypothesized to exist somewhere between SMA-negative and positive fibroblasts, and named a "proto-myofibroblast". The sub-epithelial spindle cells of normal and malignant tumors of the GI, GU, and respiratory tracts are all fibroblasts with SFs. The second erroneous myofibroblast came from a 1971 rat wound healing study and its 1974 human counterpart. Updated analysis of the papers' TEMs proved that the cells are mSMCs and not fibroblasts (AKA: MFs). The pathognomonic cells of Dupuytren's contracture are mSMCs and fibroblasts and that of the stenosing arteriopathy of Kawasaki Disease and other similar arteriopathies are mSMCs. TEM remains a powerful tool.