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Tumor acidic microenvironment is the main feature of many solid tumors. As a part of the tumor microenvironment, it has a profound impact on the occurrence and development of tumors. However, the research on how tumor cells sense the changes of the external microenvironment and how the intracellular subcellular structures transmit the signals from extracellular to intracellular is unclear. In this study, we identify that the acidic microenvironment enhances cancer cell motility, and the expression of membrane-anchored membrane type 1-matrix metalloproteinase is also associated with cell motility, which indicates more degradation of the ECM under the acidic microenvironment. Moreover, the expression of cofilin is low in the acidic microenvironment, and the F-actin filaments are distributed more along the cells. The cytoskeletal F-actin changes are consistent with the potential of a high-invasive phenotype. Further study reveals the upstream control of the signal transductions from extracellular to intracellular, that is, the integrin β1 functions to trigger the biological responses under the acidic microenvironment. Our results demonstrate that the acidic microenvironment enhances cancer cell motility through the integrin β1/cofilin/F-actin signal axis. This study clearly shows the scheme of the signal transmissions from extracellular to intracellular and further reveals the cytoskeletal roles for the contributions of cancer cell motility under acidic microenvironment, which provides new targets for cancer intervention from the biochemical and biophysical perspectives., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biomechanical cues within tissue microenvironments are critical for maintaining homeostasis, and their disruption can contribute to malignant transformation and metastasis. Once transformed, metastatic cancer cells can migrate persistently by adapting (plasticity) to changes in the local fibrous extracellular matrix, and current strategies to recapitulate persistent migration rely exclusively on the use of aligned geometries. Here, the controlled interfiber spacing in suspended crosshatch networks of nanofibers induces cells to exhibit plasticity in migratory behavior (persistent and random) and the associated cytoskeletal arrangement. At dense spacing (3 and 6 µm), unexpectedly, elongated cells migrate persistently (in 1 dimension) at high speeds in 3-dimensional shapes with thick nuclei, and short focal adhesion cluster (FAC) lengths. With increased spacing (18 and 36 µm), cells attain 2-dimensional morphologies, have flattened nuclei and longer FACs, and migrate randomly by rapidly detaching their trailing edges that strain the nuclei by ∼35%. At 54-µm spacing, kite-shaped cells become near stationary. Poorly developed filamentous actin stress fibers are found only in cells on 3-µm networks. Gene-expression profiling shows a decrease in transcriptional potential and a differential up-regulation of metabolic pathways. The consistency in observed phenotypes across cell lines supports using this platform to dissect hallmarks of plasticity in migration in vitro .-Jana, A., Nookaew, I., Singh, J., Behkam, B., Franco, A. T., Nain, A. S. Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response.

When a cell migrates, the centrosome positions between the nucleus and the leading edge of migration via the microtubule system. The protein Crp F46 (centrosome-related protein F46) has a known role during mitosis and centrosome duplication. However, how Crp F46 efficiently regulates centrosome-related cell migration is unclear. Here, we report that knockdown of Crp F46 resulted in the disruption of microtubule arrangement, with impaired centrosomal reorientation, and slowed down cell migration. In cells that express low levels of Crp F46 , stress fibers were weakened, which could be rescued by recovering Flag-Crp F46 . We also found that Crp F46 interacted with non-muscle myosin high chain IIA (NMHC IIA) and that its three coiled-coil domains are pivotal for its binding to NMHC IIA. Additionally, analyses of phosphorylation of NMHC IIA and RLC (regulatory light chain) demonstrated that Crp F46 was associated with myosin IIA during filament formation. Indirect immunofluorescence images indicated that NM IIA filaments were inhibited when Crp F46 was under-expressed. Thus, Crp F46 regulates cell migration by centrosomal reorientation and altering the function of the actomyosin network by controlling specific phosphorylation of myosin., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The actin cytoskeleton has long been recognized as a crucial sub-cellular filament system that is responsible for governing fundamental events ranging from cell division and muscle contraction to whole cell motility and the maintenance of tissue integrity. Consequently, it is not surprising that this network is the focus of over 100,000 different manuscripts. Alterations in the actin cytoskeleton lead to an assortment of diseases and serve as a target for a variety of pathogens. Here we have brought together a collection of primary research articles and reviews that underscore the broad influence this filament system has on organisms. Anat Rec, 301:1986-1990, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Our previous paper reported the structure of ginseng pectic polysaccharides related to the cell migration inhibitory effects, but the underlying mechanisms are poorly understood. In this manuscript, rhamnogalacturonan I (RGI)-rich pectins prepared from ginseng pectin were investigated for their effect on cell migration. The results indicated that the combination of homogalacturonan (HG) and RGI-rich pectins exerted stronger effects than either HG- or RGI-rich pectin alone. Further studies revealed that the effects of HG- and RGI-rich pectins were dependent on pretreatment, which caused alterations in cell morphologies such as cell size and shape, focal adhesion, and the organization of actin filaments, suggesting that HG and RGI pectins exert synergistic effects on cell migration, likely through different ways. Morphological data and quantitative cell adhesion and spreading assays showed that HG- and RGI-rich pectin treatment decreased cell adhesion and cell spreading on the substratum, suggesting that HG- and RGI-rich pectins may exert their effects on cell migration via decreasing cell adhesion and cell spreading. Additionally, we showed that L-929 cells expressed little galectin-3 (Gal-3) and that lactose, an inhibitor of Gal-3 did not block the activities of HG- and RGI-rich pectins, implicating that cell migration inhibited by pectin did not correlate to Gal-3., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8 + T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8 + T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Platelets are small anucleate blood cells involved in haemostasis. Platelet activation, caused by agonists such as thrombin or by contact with the extracellular matrix, leads to platelet adhesion, aggregation, and coagulation. Activated platelets undergo shape changes, adhere, and spread at the site of injury to form a blood clot. We investigated the morphology and morphological dynamics of human platelets after complete spreading using fast scanning ion conductance microscopy (SICM). In contrast to unstimulated platelets, thrombin-stimulated platelets showed increased morphological activity after spreading and exhibited dynamic morphological changes in the form of wave-like movements of the lamellipodium and dynamic protrusions on the platelet body. The increase in morphological activity was dependent on thrombin concentration. No increase in activity was observed following exposure to other activation agonists or during contact-induced activation. Inhibition of actin polymerization and inhibition of dynein significantly decreased the activity of thrombin-stimulated platelets. Our data suggest that these morphological dynamics after spreading are thrombin-specific and might play a role in coagulation and blood clot formation.

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration., Competing Interests: The authors declare no conflict of interest.

Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In order for the cell to function well within a multicellular system, the mechanical properties of the plasma membrane need to meet two different requirements: cell shape maintenance and rearrangement. To achieve these goals, phosphoinositides play key roles in the regulation of the cortical actin cytoskeleton. PI(4,5)P₂is the most abundant phosphoinositide species in the plasma membrane. It maintains cell shape by linking the actin cortex to the membrane via interactions with Ezrin/Radixin/Moesin (ERM) proteins and class I myosins. Although the role of D3-phosphoinositides, such as PI(3,4,5)P₃, in actin-driven cell migration has been a subject of controversy, it becomes evident that the dynamic turnover of the phosphoinositide by the action of metabolizing enzymes, such as 5-phosphatases, is necessary. Recent studies have revealed an important role of PI(3,4)P₂in podosome/invadopodia formation, shedding new light on the actin-based organization of membrane structures regulated by phosphoinositide signaling. This article is part of a Special Issue entitled Phosphoinositides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this "stiffness cation" unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Platelet-rich plasma (PRP) is a platelet concentrate in a small volume of plasma. It is highly enriched in growth factors able to stimulate the migration and growth of bone-forming cells. PRP is often used in clinical applications, as dental surgery and fracture healing. Platelet derived growth factor (PDGF), is highly concentrated in PRP and it was shown in our previous studies to provide the chemotactic stimulus to SaOS-2 osteoblasts to move in a microchemotaxis assay. Aim of the present studies is to analyze the effects of a PRP pretreatment (short time course: 30-150 min) of SaOS-2 cells with PRP on the organization of actin cytoskeleton, the main effector of cell mobility. The results indicate that a pretreatment with PRP increases chemokinesis and chemotaxis and concomitantly induces the organization of actin microfilaments, visualized by immunocytochemistry, in a directionally elongated phenotype, which is characteristic of the cells able to move. PRP also produces a transient increase in the expression of PGDF α receptor. This reorganization is blocked by the immunoneutralization of PDGF demonstrating the responsibility of this growth factor in triggering the mechanisms responsible for cellular movements.

Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Children with fetal alcohol syndrome (FAS) display striking craniofacial abnormalities. These features are proposed to result from perturbations in the morphology and function of cranial neural crest cells (cNCCs), which contribute significantly to the craniofacial complex. While certain pathways by which this may occur have been suggested, precise teratogenic mechanisms remain intensely investigated, as does the question of the teratogenic dose. The present study focused on examining how avian cNCC actin cytoskeleton, migratory distance, and proliferation are affected ex vivo by exposure to ethanol concentrations that simulate maternal intoxication. Chick cNCCs were cultured in 0.2% and 0.4% v/v ethanol. Distances migrated by both ethanol-treated and control cells at 24 and 48 h were recorded. Following phalloidin immunocytochemistry, treated and control cNCCs were compared morphologically and quantitatively. Apoptosis and proliferation in control versus treated cNCCs were also studied. Chick cNCCs cultured in ethanol lost their spindle-like shapes and their ordered cytoskeleton. There was a significant stage-dependent effect on cNCC migration at 24 h (p = 0.035), which was greatest at stage 10 (HH). Ethanol treatment for 48 h revealed a significant main effect for ethanol, chiefly at the 0.4% level. There was also an interaction effect between ethanol dose and stage of development (stage 9 HH). Actin microfilament disruption was quantitatively increased by ethanol at the doses studied while cNCC proliferation was increased but not significantly. Ethanol had no effect on cNCC apoptosis. At ethanol levels likely to induce human FAS, avian cNCCs exhibit various subtle, potentially significant changes in morphology, migration, and proliferation, with possible consequences for fated structures., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Focal adhesions (FAs) are complex plasma membrane-associated macromolecular assemblies that serve to physically connect the actin cytoskeleton to integrins that engage with the surrounding extracellular matrix (ECM). FAs undergo maturation wherein they grow and change composition differentially to provide traction and to transduce the signals that drive cell migration, which is crucial to various biological processes, including development, wound healing and cancer metastasis. FA-related signalling networks dynamically modulate the strength of the linkage between integrin and actin and control the organization of the actin cytoskeleton. In this review, we have summarized a number of recent investigations exploring how FA composition is affected by the mechanical forces that transduce signalling networks to modulate cellular function and drive cell migration. Understanding the fundamental mechanisms of how force governs adhesion signalling provides insights that will allow the manipulation of cell migration and help to control migration-related human diseases., (© 2013 The Authors Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our in vitro model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which "opens the door" in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.

The aim of this article is to study cell deformation and cell movement by considering both the mechanical and biochemical properties of the cortical network of actin filaments and its concentration. Actin is a polymer that can exist either in filamentous form (F-actin) or in monometric form (G-actin) (Chen et al. in Trends Biochem Sci 25:19-23, 2000) and the filamentous form is arranged in a paired helix of two protofilaments (Ananthakrishnan et al. in Recent Res Devel Biophys 5:39-69, 2006). By assuming that cell deformations are a result of the cortical actin dynamics in the cell cytoskeleton, we consider a continuum mathematical model that couples the mechanics of the network of actin filaments with its bio-chemical dynamics. Numerical treatment of the model is carried out using the moving grid finite element method (Madzvamuse et al. in J Comput Phys 190:478-500, 2003). Furthermore, by assuming slow deformations of the cell, we use linear stability theory to validate the numerical simulation results close to bifurcation points. Far from bifurcation points, we show that the mathematical model is able to describe the complex cell deformations typically observed in experimental results. Our numerical results illustrate cell expansion, cell contraction, cell translation and cell relocation as well as cell protrusions. In all these results, the contractile tonicity formed by the association of actin filaments to the myosin II motor proteins is identified as a key bifurcation parameter.

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.

Migration of motile cells on flat substrates is usually driven by the polymerization of a flat actin filament network. Theoretical models have made different predictions regarding the distribution of the filament orientation in the lamellipodium with respect to the direction of motion. Here we show how one can automatically reconstruct the orientation distribution of actin filaments in the lamellipodium of migrating keratocytes from electron microscopy tomography data. We use two different image analysis methods, an algorithm which explicitly extracts an abstract network representation and an analysis of the gray scale information based on the structure tensor. We show that the two approaches give similar results, both for simulated data and for electron microscopy tomography data from migrating keratocytes. For the lamellipodium at the leading edge of fast moving cells, we find an orientation distribution that is peaked at +35/-35 degrees. For the lamellipodium at the leading edge of slow moving cells as well as for the lamellipodium at the flanks of fast moving cells, one broad peak around 0 degree dominates the distribution., (Copyright © 2012 International Society for Advancement of Cytometry.)

Changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes during their migration in fish, frogs and birds have been studied by the method of confocal laser scanning microscopy. It has been shown that, during movement of cells, the reorganization of cytoskeleton microfilaments in erythrocytes is similar to that in leukocytes. During migration, red blood cells of amphibious and birds form pseudopodia filled with bunches in parallel laid actin filaments. Erythrocytes in fish do not form pseudopodia. Similar to leukocytes change in the structure of the actin cytoskeleton in nuclear erythrocytes determines the ability of red blood cells to reactions of migration and phagocytosis.

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

Background: Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton., Methodology/principal Findings: We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I., Conclusions/significance: Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway.

During cell migration, forces generated by the actin cytoskeleton are transmitted through adhesion complexes to the substrate. To investigate the mechanism of force generation and transmission, we analyzed the relationship between actin network velocity and traction forces at the substrate in a model system of persistently migrating fish epidermal keratocytes. Front and lateral sides of the cell exhibited much stronger coupling between actin motion and traction forces than the trailing cell body. Further analysis of the traction-velocity relationship suggested that the force transmission mechanisms were different in different cell regions: at the front, traction was generated by a gripping of the actin network to the substrate, whereas at the sides and back, it was produced by the network's slipping over the substrate. Treatment with inhibitors of the actin-myosin system demonstrated that the cell body translocation could be powered by either of the two different processes, actomyosin contraction or actin assembly, with the former associated with significantly larger traction forces than the latter.

Cell polarity proteins regulate tight junction formation and directional migration in epithelial cells. To date, the mechanism by which these polarity proteins assemble at the leading edge of migrating epithelial cells remains unclear. We report that occludin, a transmembrane protein, is localized at the leading edge of migrating cells and regulates directional cell migration. During migration, occludin knockdown disrupted accumulation of aPKC-Par3 and PATJ at the leading edge, and led to a disorganized microtubule network and defective reorientation of the microtubule organization center (MTOC). Phosphorylation of occludin at tyrosine 473 residue allowed recruitment of p85 alpha to the leading edge via association with its C-terminal SH2 domain. Loss of occludin attenuated activation of PI3K, leading to disorganization of the actin cytoskeleton and reduced cell protrusions. Our data indicate that occludin is required for the leading-edge localization of polarity proteins aPKC-Par3 and PATJ and promotes cell protrusion by regulating membrane-localized activation of PI3K., ((c) 2010 Elsevier Inc. All rights reserved.)

The protein actin forms filaments that provide cells with mechanical support and driving forces for movement. Actin contributes to biological processes such as sensing environmental forces, internalizing membrane vesicles, moving over surfaces, and dividing the cell in two. These cellular activities are complex; they depend on interactions of actin monomers and filaments with numerous other proteins. Here, we present a summary of the key questions in the field and suggest how those questions might be answered. Understanding actin-based biological phenomena will depend on identifying the participating molecules and defining their molecular mechanisms. Comparisons of quantitative measurements of reactions in live cells with computer simulations of mathematical models will also help generate meaningful insights.

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.

Structural remodeling of neurons occurs during development, regeneration, and in relation to learning and memory acquisition processes. These processes are associated with reconfiguration of the cytoskeleton, and are accompanied by adjustments of the plasma membrane surface area. Using cultured Aplysia neurons, we examined the role of membrane budgeting and the submembrane actin skeleton in determining the dimensions and shape of neurons. We found that the dimensions and cytoarchitecture of cultured neurons reach a steady state by maintaining a balance between constitutive plasma membrane retrieval and insertion. Constitutive membrane retrieval and the addition of Golgi-derived membranes are not coupled. Therefore, inhibition of Golgi-derived membrane supply by brefeldin A (BFA) leads to total retraction of the neurites. The process of BFA-induced neurite retraction is reversible upon washout. BFA-induced neurite retraction is associated with orderly retrograde shortening of the microtubule (MT) skeleton. The actin-perturbing drugs cytochalasin B, latrunculin A, or jasplakinolide block constitutive membrane retrieval. In the presence of the actin-perturbing drugs, depletion of Golgi-derived membrane supply by BFA does not lead to neurite retraction. Taken together, the present study reveals that the ratio of constitutive plasma membrane retrieval/exocytosis plays a pivotal role in defining the neuron's dimensions and cytoarchitecture. Submembrane actin dynamics plays a key role in regulating the rates of constitutive membrane retrieval. We propose that the coordination between MT polymerization and the surface area of the plasma membrane is coordinated by a linking mechanism in which the submembrane actin plays a major role.

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibrils and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced a more than 1.5-fold accumulation of stress fibrils only in control cells but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type but not in cells with a low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, beta actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues.

Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically up-regulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer.

Gene regulatory networks direct the progressive determination of cell fate during embryogenesis, but how they control cell behavior during morphogenesis remains largely elusive. Cell sorting, microarrays, and targeted molecular manipulations were used to analyze cardiac cell migration in the ascidian Ciona intestinalis. The heart network regulates genes involved in most cellular activities required for migration, including adhesion, cell polarity, and membrane protrusions. We demonstrated that fibroblast growth factor signaling and the forkhead transcription factor FoxF directly upregulate the small guanosine triphosphatase RhoDF, which synergizes with Cdc42 to contribute to the protrusive activity of migrating cells. Moreover, RhoDF induces membrane protrusions independently of other cellular activities required for migration. We propose that transcription regulation of specific effector genes determines the coordinated deployment of discrete cellular modules underlying migration.

Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at > or = 30 microM inhibited anterior redistribution of myosin IIA, with 100 microM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t(1/2) < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453-465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of approximately 10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with approximately 100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics.

Posttranslational arginylation is critical for mouse embryogenesis, cardiovascular development, and angiogenesis, but its molecular effects and the identity of proteins arginylated in vivo are unknown. We found that beta-actin was arginylated in vivo to regulate actin filament properties, beta-actin localization, and lamella formation in motile cells. Arginylation of beta-actin apparently represents a critical step in the actin N-terminal processing needed for actin functioning in vivo. Thus, posttranslational arginylation of a single protein target can regulate its intracellular function, inducing global changes on the cellular level, and may contribute to cardiovascular development and angiogenesis.

Podosomes, highly dynamic adhesion structures implicated in cell motility and extracellular matrix degradation, are characteristic of certain cells of the myeloid lineage and a limited range of other cell types. The nature and the mechanisms that regulate their high turnover are unknown at present. The cysteine protease calpain is involved in the regulation of cell migration in part by promoting either formation or disassembly of adhesion sites. Despite the fact that many known substrates of calpain are also structural components of the podosome complex, no studies have yet demonstrated that calpain participates in the regulation of podosome dynamics. In the present work, we show that inhibition of calpain in primary mouse dendritic cells leads to enhanced accumulation of actin filaments, the Wiskott Aldrich Syndrome protein (WASP), beta(2) integrins, talin, paxillin and vinculin in podosomes. This accumulation of components is associated with stabilisation of podosome turnover, overall reduction in velocity of cell locomotion and impaired transmigration across an endothelial monolayer. We also demonstrate that calpain cleaves the podosome components talin, Pyk2 and WASP in dendritic cells. In summary, our results provide evidence that calpain regulates podosome composition and turnover and that this process is required for efficient migration of dendritic cells.

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling., (Copyright 2004 Wiley-Liss, Inc.)

During epithelial cell migration, membrane ruffles can be visualized by phase contrast microscopy as dark waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body. Despite the common use of the term membrane ruffles, their structure, molecular composition, and the mechanisms leading to their formation remained largely unknown. We show here that membrane ruffles differ from the underlying cell lamella by more densely packed bundles of actin filaments that are enriched in the actin cross-linkers filamin and ezrin, pointing to a specific bundling process based on these cross-linkers. The accumulation of phosphorylated, that is, inactivated, cofilin in membrane ruffles suggests that they are compartments of inhibited actin filament turnover. High Rac1 and low RhoA activities were found under conditions of suboptimal integrin-ligand interaction correlating with low lamellipodia persistence, inefficient migration, and high ruffling rates. Based on these findings, we define membrane ruffles as distinct compartments of specific composition that form as a consequence of inefficient lamellipodia adhesion.

The rearrangement of the actin cytoskeleton has been shown to play a critical role in the development of transformation and malignant phenotype of cancer cells. Rho family GTPases regulate the arrangement of the actin cytoskeleton. By wound-healing assay, we have found that NIH 3T3 fibroblast cells move towards the wound- gaps by extending filopodial and lamellipdial structures at the leading edge of the moving cells. We have inactivated the function of Rho GTPases of v-Ras transformed NIH 3T3 cells by overexpressing Rho GTPase-activating (RhoGAP) domain of RhoGAP of p190. We have observed that inactivation of Rho, Rac and Cdc42 GTPases by overexpressing RHG causes inhibition of: (i) polymerization of actin to form filaments, (ii) formation of lamellipodia, filopodia and stress fibres, (iii) cell motility, (iv) cell spreading and (v) cell-to-cell adhesions. These results further strengthen the current knowledge on the role of Rho, Rac and Cdc42 GTPases in the regulation of the rearrangement of actin cytoskeleton. Our results, for the first time, demonstrate that RhoGAP domain of RhoGAP could be used to study the molecular mechanism of Ras-mediated signalling in growth, differentiation and carcinogenesis.

Curcumin is a dietary phytochemical associated with anti-tumorigenic effects, but the mechanisms by which it inhibits cancer cell growth and metastasis are not completely understood. For example, little information is available regarding the effects of curcumin on cytoskeletal organization and function. In this study, time-lapse video and immunofluorescence labeling methods were used to demonstrate that curcumin significantly alters microfilament organization and cell motility in PC-3 and LNCaP human prostate cancer cells in vitro. Curcumin rapidly arrests cell movements and subsequently alters cell shape in the highly motile PC-3 cell line, but has a less noticeable effect on the relatively immobile LNCaP cell line. Stress fibers are augmented, and the overall quantity of f-actin appears to increase in both types of cells following curcumin treatment. Cytochalasin B (CB) disrupts microfilament organization in both cell lines, and causes vigorous membrane blebbing in PC-3 cells, but not LNCaP cells. Pre-treatment of cells with curcumin suppresses changes in microfilament organization caused by CB, and blocks PC-3 membrane blebbing. At least some of the effects of curcumin appear to be mediated by protein kinase C (PKC), as treatment with the PKC inhibitor bisindolylmaleimide inhibits the ability of curcumin to block CB-induced membrane blebbing. These findings demonstrate that curcumin exerts significant effects on the actin cytoskeleton in prostate cancer cells, including altering microfilament organization and function. This is a novel observation that may represent an important mechanism by which curcumin functions as a chemopreventative agent, and as an inhibitor of angiogenesis and metastasis., (Copyright 2004 Wiley-Liss, Inc.)

Doublecortin is a microtubule-associated protein that is essential for normal brain development. A recent report published in Molecular Cell shows that doublecortin associates preferentially with microtubules made of 13 protofilaments, by recognizing a novel site between the protofilaments. These findings explain how doublecortin stabilizes microtubules and provide clues about its function during neuronal migration.

Motile fibroblasts generate forces that can be expressed as cell migration or as traction, the drawing-in of extracellular matrix. Traction by cultured fibroblasts can induce a rapid concerted reorganization of collagen gel, creating a pattern of collagen alignment similar to that seen in tendons and ligaments. In such fibrous connective tissues, after pattern morphogenesis is complete, ongoing traction may be responsible for the maintenance of proper form and function. The molecules that generate and transmit forces have been catalogued; however, how these nanometer-scale molecules contribute to millimeter-scale patterns has not been directly tested. Here, we placed pairs of explants of human periodontal ligament fibroblasts in collagen gels, where ligament-like straps of anisotropic collagen formed on the axes between them. We scrutinized the traction apparatus using electron microscopy, video microscopy, and computer-based pattern analysis, augmented with pharmacologic inhibitors of cytoskeletal function. Patterning was marked by the co-alignment of collagen, fibroblasts, and their actin cytoskeletons, all parallel to the axis between explants. The pattern was diminished by depolymerizing actin filaments or by blocking myosin activity, but was accentuated by depolymerizing microtubules. The plasma membrane also seems to contribute to the traction force. These molecular components combine to exert a sub-maximal traction force on the matrix, suggesting that the force may be regulated to ensure tissue tensional homeostasis., (Copyright 2004 Wiley-Liss, Inc.)

Two types of sperm are produced in the silkworm, Bombyx mori. Nucleate eupyrene sperm is an ordinary sperm that contributes to fertilization, while anucleate apyrene sperm is considered to play important roles in assisting eupyrene sperm. At the very late stage of spermatogenesis, a phenomenon called "peristaltic squeezing" occurs in both types of sperm, whereby cytoplasm of the eupyrene and nuclei of the apyrene sperm are discarded from the posterior end, forming matured sperm. In this study, rhodamine-phalloidin staining for actin was applied to sperm bundles. Before the start of peristaltic squeezing, actin filament networks are spread on the cyst cells and constrictions by the networks appear in several places of the bundles. Actin particles, which are later recognized as circlets, are localized within the bundles. Squeezing action by the networks occurs from the anterior region and transfers toward the posterior, eliminating cytoplasm together with circlets from the posterior end. It seems that actin filaments contribute to the peristaltic squeezing of the sperm bundles in Bombyx mori., (Copyright 2003 Wiley-Liss, Inc.)