Actomyosin Contraction Research Articles

Effects of Stretch on the Bladder Umbrella Cell Apical Junctional Complex

The urothelium, which lines the luminal surface of the bladder, must maintain its integrity during cycles of filling and voiding. How this is accomplished is not well understood. A critical component of the epithelial barrier is the apical junctional complex (AJC), which is a ring‐like structure circumscribing polarized epithelial cells at their apical pole. The AJC includes the apical‐most tight junction (TJ), subjacent adherens junction (AJ), and associated actin cytoskeleton, which modulates cell tension at regions of cell‐cell contact. We previously reported that the TJ ring encircling the outermost umbrella cell layer of the urothelium expands during bladder filling and contracts during voiding. However, whether the AJ is similarly affected, and the identity of the underlying machinery that drives these changes in the AJC remains unknown.When we examined bladders that were filled and voided in situ we observed the following: 1) Like the TJ ring, the AJ ring also expanded during filling and contracted within minutes of voiding. 2) In addition to actin, which was concentrated in a continuous ring interposed between the TJ and AJ, the actin bundling protein a‐actinin‐4, and non‐muscle myosin IIA, which regulates AJ dynamics, also associated with the AJC ring. 3) The actin disrupting agent cytochalasin D, as well as inhibitors of formins (SMIFH2) or Arp2/3 (CK869), decreased expansion of the AJC ring indicating a role for active actin polymerization in this process. Surprisingly, treatment with blebbistatin, a myosin II contraction inhibitor, had no effect on the expansion of the AJC ring. 4) An exocytosis inhibitor, brefeldin A, showed a strong inhibitory effect on AJC ring expansion, indicating that vesicular traffic may regulate AJC dynamics. 5) To target the exocytic machinery more specifically we expressed dominant‐negative (DN) RAB8A, RAB11A, or RAB13 mutants. Whereas DN‐RAB13, previously implicated in the regulation of TJ dynamics, significantly decreased AJC ring expansion during bladder filling, DN‐RAB8A and DN‐RAB11A, which are involved in stretch‐stimulated exocytosis during bladder filling, did not. 6) Unlike expansion of the AJC ring, its voiding‐induced contraction was strongly inhibited by blebbistatin and was also significantly inhibited by cytochalasin D. 7) Intriguingly, AJC ring contraction was also inhibited by the general endocytosis inhibitor Pitstop2 or expression of a DN mutant of the endocytic regulatory protein Dynamin2. Moreover, DN‐RhoA, which we previously implicated in umbrella cell apical endocytosis, similarly impaired AJC ring contraction.ConclusionsOur studies indicate that a likely mechanism for retention of urothelial continuity in the face of cyclical changes in volume is expansion and contraction of the AJC ring. Not only does AJC ring expansion depend on actin polymerization, it also requires exocytosis, presumably of vesicles containing AJC‐associated membrane proteins. In contrast, contraction of the AJC ring is likely mediated by actomyosin contraction of the junctional ring and endocytosis of excess AJC proteins.Support or Funding Information1F31DK117547‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Force Transmission between Three Tissues Controls Bipolar Planar Polarity Establishment and Morphogenesis

How tissues from different developmental origins interact to achieve coordinated morphogenesis at the level of a whole organism is a fundamental question in developmental biology. While biochemical signaling pathways controlling morphogenesis have been extensively studied [1-3], morphogenesis of epithelial tissues can also be directed by mechanotransduction pathways physically linking two tissues [4-8]. C.elegans embryonic elongation requires the coordination of three tissues: muscles, the dorsal and ventral epidermis, and the lateral epidermis. Elongation starts by cell-shape changes driven by actomyosin contractions in the lateral epidermis [9,10]. At mid-elongation, muscles become connected to the apical surface of the dorsal and ventral epidermis by molecular tendons formed by muscle integrins, extracellular matrix, and C.elegans hemidesmosomes (CeHDs). The mechanical signal generated by the onset of muscle contractions in the antero-posterior axis from mid-elongation is translated into a biochemical pathway controlling the maturation of CeHDs in the dorsal and ventral epidermis [11]. Consistently, mutations affecting muscle contractions or molecular tendons lead to a mid-elongation arrest [12]. Here, we found that the mechanical force generated by muscle contractions and relayed by molecular tendons is transmitted by adherens junctions to lateral epidermal cells, where it establishes a newly identified bipolar planar polarity of the apical PAR module. The planar polarized PAR module is then required for actin planar organization, thus contributing to the determination of the orientation of cell-shape changes and the elongation axis of the whole embryo. This mechanotransduction pathway is therefore essential to coordinate the morphogenesis of three embryonic tissues.

Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism

Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid biosynthesis and adipogenesis by controlling the expression of several enzymes required for cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In vertebrates, SREBP activation is mainly controlled by a complex and well-characterized feedback mechanism mediated by cholesterol, a crucial bio-product of the SREBP-activated mevalonate pathway. In this work, we identified acto-myosin contractility and mechanical forces imposed by the extracellular matrix (ECM) as SREBP1 regulators. SREBP1 control by mechanical cues depends on geranylgeranyl pyrophosphate, another key bio-product of the mevalonate pathway, and impacts on stem cell fate in mouse and on fat storage in Drosophila. Mechanistically, we show that activation of AMP-activated protein kinase (AMPK) by ECM stiffening and geranylgeranylated RhoA-dependent acto-myosin contraction inhibits SREBP1 activation. Our results unveil an unpredicted and evolutionary conserved role of SREBP1 in rewiring cell metabolism in response to mechanical cues.

Regulation of Actin Dynamics in the C. elegans Somatic Gonad.

The reproductive system of the hermaphroditic nematode C. elegans consists of a series of contractile cell types—including the gonadal sheath cells, the spermathecal cells and the spermatheca–uterine valve—that contract in a coordinated manner to regulate oocyte entry and exit of the fertilized embryo into the uterus. Contraction is driven by acto-myosin contraction and relies on the development and maintenance of specialized acto-myosin networks in each cell type. Study of this system has revealed insights into the regulation of acto-myosin network assembly and contractility in vivo.

Computational modeling of epithelial wound healing: Short and long term chemo-mechanical mechanisms

During the lifetime of all living multicellular organisms, wounds in their tissues are frequently observed. The capability of closing those gaps is fundamental for a healthy development. If done deficiently, many diseases may occur from simple inflammation to tumor formation. The wound healing process in epithelial tissue occurs in three different stages. The first one is the assembly of a supra-cellular actomyosin cable and its migration towards the wound edge, triggered by biochemical processes in which calcium plays a distinctive role. How this process is orchestrated following damage remains unclear. Later, after its positioning, the cable contracts driving the tissue towards the gap and reducing the wound area. Finally, cell migration towards the interior of the wound ends up sealing the tissue. In this work, we make use of a mechanical continuum model for the first two stages in order to developed and 2D finite element simulations within a monolithically fully implicit implementation. The model for the actomyosin cable formation involves the coupling of transient calcium ions transport, with actin fibers and myosin motors recruitment and non-linear mechanical response of the tissue. The contraction stage, the active deformation of the previously formed actomyosin cable is taken into account. The relative motion of the myosin motors over the actin filaments is modeled so there exists an active tissue contraction in the direction of those fibers. Upon implementation, the model is capable of performing a wide range of biophysical situations reported experimentally, as we demonstrate in our numerical results. We have been able to rationalize through computational mechanics the firing of calcium in the wound right after damage infliction as well as the consequent formation of actin ring, reproducing nicely what has been reported in biological literature. Thereafter, the numerical model of acto-myosin contraction, fully integrated with the non-linear mechanics of the problem, correlates with the mechanics of wound closure at the actin-ring contraction stage. More importantly, the approach is the first of its kind in the modeling of epithelial and embryonic cell layers, where a wide number of complex mechanics has been integrated and solved though computational methods in engineering. We believe that the simulations will help to unravel new insights in open questions of developmental biology.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA - /E -/H - and racE - mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

ZnUMBA Crosses the Border

Breaks in tight junctions cause transient tissue leaks. In this issue of Developmental Cell, Stephenson etal. (2019) show that local RhoA activation and actomyosin contractions concentrate tight junction proteins at the breach, which repairs the leak.

PCP-dependent transcellular regulation of actomyosin oscillation facilitates convergent extension of vertebrate tissue

Oscillatory flows of actomyosin play a key role in the migration of single cells in culture and in collective cell movements in Drosophila embryos. In vertebrate embryos undergoing convergent extension (CE), the Planar Cell Polarity (PCP) pathway drives the elongation of the body axis and shapes the central nervous system, and mutations of the PCP genes predispose humans to various malformations including neural tube defects. However, the spatiotemporal patterns of oscillatory actomyosin contractions during vertebrate CE and how they are controlled by the PCP signaling remain unknown. Here, we address these outstanding issues using a combination of in vivo imaging and mathematical modeling. We find that effective execution of CE requires alternative oscillations of cortical actomyosin across cell membranes of neighboring cells within an optimal frequency range. Intriguingly, temporal and spatial clustering of the core PCP protein Prickle 2 (Pk2) is correlated to submembranous accumulations of F-actin, and depletion of Pk2 perturbs the oscillation of actomyosin contractions. These findings shed light on the significance of temporal regulation of actomyosin contraction by the PCP pathway during CE, in addition to its well-studied spatial aspects.

Micro-scaled topographies direct differentiation of human epidermal stem cells

Human epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. Statement of SignificanceThe epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.

Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells.

SummaryNicotinamide, the amide form of vitamin B3, is widely used in disease treatments and stem cell applications. However, nicotinamide's impact often cannot be attributed to its nutritional functions. In a vitamin screen, we find that nicotinamide promotes cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibits the phosphorylation of myosin light chain, suppresses actomyosin contraction, and leads to improved cell survival after individualization. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and casein kinase 1. We demonstrate that nicotinamide affects human embryonic stem cell pluripotency and differentiation as a selective kinase inhibitor. The findings in this report may help researchers design better strategies to develop nicotinamide-related stem cell applications and disease treatments.

Shear-induced damped oscillations in an epithelium depend on actomyosin contraction and E-cadherin cell adhesion.

Shear forces between cells occur during global changes in multicellular organization during morphogenesis and tissue growth, yet how cells sense shear forces and propagate a response across a tissue is unknown. We found that applying exogenous shear at the midline of an epithelium induced a local, short-term deformation near the shear plane, and a long-term collective oscillatory movement across the epithelium that spread from the shear-plane and gradually dampened. Inhibiting actomyosin contraction or E-cadherin trans-cell adhesion blocked oscillations, whereas stabilizing actin filaments prolonged oscillations. Combining these data with a model of epithelium mechanics supports a mechanism involving the generation of a shear-induced mechanical event at the shear plane which is then relayed across the epithelium by actomyosin contraction linked through E-cadherin. This causes an imbalance of forces in the epithelium, which is gradually dissipated through oscillatory cell movements and actin filament turnover to restore the force balance across the epithelium.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.

Selective Laminin-Directed Differentiation of Human Induced Pluripotent Stem Cells into Distinct Ocular Lineages

The extracellular matrix plays a key role in stem cell maintenance, expansion, and differentiation. Laminin, a basement membrane protein, is a widely used substrate for cell culture including the growth of human induced pluripotent stem cells (hiPSCs). Here, we show that different isoforms of laminin lead to the selective differentiation of hiPSCs into different eye-like tissues. Specifically, the 211 isoform of the E8 fragment of laminin (LN211E8) promotes differentiation into neural crest cells via Wnt activation, whereas LN332E8 promotes differentiation into corneal epithelial cells. The immunohistochemical distributions of these laminin isoforms in the developing mouse eye mirrors the hiPSC type that was induced invitro. Moreover, LN511E8 enables generation of dense hiPSC colonies due to actomyosin contraction, which in turn led to cell density-dependent YAP inactivation and subsequent retinal differentiation in colony centers. Thus, distinct laminin isoforms determine the fate of expanded hiPSCs into eye-like tissues.

αE-catenin is a candidate tumor suppressor for the development of E-cadherin-expressing lobular-type breast cancer.

Although mutational inactivation of E‐cadherin (CDH1) is the main driver of invasive lobular breast cancer (ILC), approximately 10–15% of all ILCs retain membrane‐localized E‐cadherin despite the presence of an apparent non‐cohesive and invasive lobular growth pattern. Given that ILC is dependent on constitutive actomyosin contraction for tumor development and progression, we used a combination of cell systems and in vivo experiments to investigate the consequences of α‐catenin (CTNNA1) loss in the regulation of anchorage independence of non‐invasive breast carcinoma. We found that inactivating somatic CTNNA1 mutations in human breast cancer correlated with lobular and mixed ducto‐lobular phenotypes. Further, inducible loss of α‐catenin in mouse and human E‐cadherin‐expressing breast cancer cells led to atypical localization of E‐cadherin, a rounded cell morphology, and anoikis resistance. Pharmacological inhibition experiments subsequently revealed that, similar to E‐cadherin‐mutant ILC, anoikis resistance induced by α‐catenin loss was dependent on Rho/Rock‐dependent actomyosin contractility. Finally, using a transplantation‐based conditional mouse model, we demonstrate that inducible inactivation of α‐catenin instigates acquisition of lobular features and invasive behavior. We therefore suggest that α‐catenin represents a bona fide tumor suppressor for the development of lobular‐type breast cancer and as such provides an alternative event to E‐cadherin inactivation, adherens junction (AJ) dysfunction, and subsequent constitutive actomyosin contraction. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

Collision of Expanding Actin Caps with Actomyosin Borders for Cortical Bending and Mitotic Rounding in a Syncytium

The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. We investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.

Coupling mechanical tension and GTPase signaling to generate cell and tissue dynamics

Regulators of the actin cytoskeleton such Rho GTPases can modulate forces developed in cells by promoting actomyosin contraction. At the same time, through mechanosensing, tension is known to affect the activity of Rho GTPases. What happens when these effects act in concert? Using a minimal model (1 GTPase coupled to a Kelvin–Voigt element), we show that two-way feedback between signaling (‘RhoA’) and mechanical tension (stretching) leads to a spectrum of cell behaviors, including contracted or relaxed cells, and cells that oscillate between these extremes. When such ‘model cells’ are connected to one another in a row or in a 2D sheet (‘epithelium’), we observe waves of contraction/relaxation and GTPase activity sweeping through the tissue. The minimal model lends itself to full bifurcation analysis, and suggests a mechanism that explains behavior observed in the context of development and collective cell behavior.

New Rock Inhibitors Action Analysis in the Cytoskeleton and Cell Migration of Tumor Cell Line MDA‐MB 231

Many patients with metastatic tumors have substantially worse survival in comparison with those patients with nonmetastatic tumors. The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis and these events became targets for the development of new antimetastatic drugs. Rho kinases, ROCK1 and ROCK2, are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. In this regard our group synthesized two sulfonylhydrazones, analogous to Fasudil (a classic Rho kinase inhibitor), named LASSBio 2020 and LASSBio 2065, differing each other by the presence of one methyl in LASSBio 2065. Therefore, the aim of the present work was to investigate the effects of these analogues against human breast carcinoma (MDA‐MB‐231) cell line migration and signalling pathways involved in in this phenomenon as well as cytoskeleton changes.For the study, cells were incubated with LASSBio 2020 or LASSBio 2065 (0.1 to 30 μM) for 24 or 48 hours. The cell viability was evaluated by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐difenltetrazolium) and plasma membrane integrity assay (lactate desidrogenase, LDH). Migration capacity of cells was evaluated using wound healing assay. Morphological analysis was carried out by phase‐contrast micrography, and analysed using Image J software. Actin structures were visualized by fluorescent phalloidin. Statistical analyses were performed by ANOVA with Newman‐Keuls post‐test (*p<0.05).The results showed that none of analogues altered viability of MDA‐MB 231 cells. The effect of the treatment with 10 and 30 μM of LASSBio 2020 decreased cell migration in 30,38% and 32,7%, respectively, when compared to control. LASSBio 2065 decreased cell migration in 24,51% (10 μM) and 26,14% (30 μM), when compared to control. Control cells presented a sprawling appearance with extended cytoplasm and lamelipodios formation. Cells incubated with 10 μm of LASSBio 2020 and LASSBio 2065 presented area, height and width reduced by 40%, membranes with filopodia, protrusions and a fusiform appearance. The staining for F‐actin showed that the cell body became elongated, less spread and with a narrowing of the cell body shape (figure 1).We can conclude that none of analogues (LASSBio 2020 and LASSBio 2065) reduced cell viability. However, the analogues reduced cell migration, altered cell morphology and interfered with formation of actin filaments. Taken together, our data can suggest that the fasudil analogues are a potential inhibitors of metastasis formation.Support or Funding InformationCapes, FAPERJ and CNPqThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defining the Role of Protein Interactions at WRAMP Structures in Directional Migration

Cell migration is a critical process in development, tissue regeneration, and cancer cell invasion, but mechanisms that establish the location of the cell rear and activate rear contractile forces are still poorly understood. Our lab previously discovered a protein network named the “Wnt5a‐receptor‐actomyosin‐polarity (WRAMP) structure” which may serve as a novel mechanism for rear‐directed control of migration. The WRAMP structure forms transiently in response to the signaling ligand Wnt5a and is characterized by polarization of F‐actin, myosin‐IIB, and melanoma cell adhesion molecule (MCAM). Cells with WRAMP structures migrate in a persistent direction for longer periods of time compared to cells without WRAMP structures, and the location where WRAMP structures form may determine the position of the cell rear and direction of migration. However, the specific interactions of proteins at WRAMP structures that activate actomyosin contraction are unknown. To address this question, we have generated plasmids to express previously identified WRAMP structure members MCAM and moesin fused to the engineered peroxidase APEX2 or the promiscuous biotin ligase BirA(R118G) for use in the proteomics techniques APEX and BioID, respectively. In these methods, proteins in close proximity to the bait protein are biotinylated and subsequently isolated on streptavidin beads for identification by mass spectrometry. Ongoing goals of this project are to compare APEX and BioID methods in melanoma cells to build up a WRAMP structure “interactome,” prioritized by interactions that increase in the presence of Wnt5a treatment. In addition, we are examining the role of RhoA/Rho kinase (ROCK) signaling in activation of actomyosin contraction using immunofluorescence for activated substrates of ROCK at WRAMP structures, and we will test a literature‐based model for RhoA activation through MCAM‐moesin interactions using an MCAM mutant which lacks moesin binding. These findings will help define the spatial organization of WRAMP structures and identify key interactions and signaling pathways involved in control of cell migration.Support or Funding InformationThis work is supported by funding from the National Institutes of Health (R01GM105997 and T32GM008759).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.