Actin-myosin Contractility Research Articles

MECHANICAL REGULATION OF MATRIX REMODELLING IN TENDON WOUND HEALING

Extracellular matrix (ECM) mechanical cues guide healing in tendons. Yet, the molecular mechanisms orchestrating the healing processes remain elusive. Appropriate tissue tension is essential for tendon homeostasis and tissue health. By mapping the attainment of tensional homeostasis, we aim to understand how ECM tension regulates healing. We hypothesize that diseased tendon returns to homeostasis only after the cells reach a mechanically gated exit from wound healing.We engineered a 3D mechano-culture system to create tendon-like constructs by embedding patient-derived tendon cells into a collagen I hydrogel. Casting the hydrogel between posts anchored in silicone allowed adjusting the post stiffness. Under this static mechanical stimulation, cells remodel the (unorganized) collagen representing wound healing mechanisms. We quantified tissue-level forces using post deflection measurements. Secreted ECM was visualized by metabolic labelling with non-canonical amino acids, click chemistry and confocal microscopy. We blocked cell-mediated actin-myosin contractility using a ROCK inhibitor (Y27632) to explore the involvement of the Rho/ROCK pathway in tension regulation.Tissue tension forces reached the same homeostatic level at day 21 independent of post compliance (p = 0.9456). While minimal matrix was synthesized in early phases of tissue formation (d3-d5), cell-deposited ECM was present in later stages (d7-d9). More ECM was deposited by tendon constructs cultured on compliant (1Nm) compared to rigid posts (p = 0.0017). Matrix synthesized by constructs cultured on compliant posts was less aligned (greater fiber dispersion, p = 0.0021). ROCK inhibition significantly decreased tissue-level tensional forces (p < 0.0001).Our results indicate that tendon cells balance matrix remodeling and synthesis during tissue repair to reach an intrinsically defined “mechanostat setpoint” guiding tension-mediated exit from wound healing towards homeostasis. We are identifying specific molecular mechanosensors governing tension-regulated healing in tendon and investigate the Rho/ROCK system as their possible downstream pathway.

Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes

Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

Abstract 2461: The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization

Abstract The motuporamines are a promising class of anti-metastatic compounds. Dihydromotuporamine C (Motu33) has been shown to activate the small GTPase RhoA, however, little is known about subsequent downstream events leading to cell migration inhibition. In the present study, we investigated the mechanism of action of Motu33 and a synthetic derivative, Motu-(CH2)-33, in Drosophila by manipulating the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity, confirming that RhoA/Rho1 is targeted by these compounds. Actin-myosin contraction is controlled by the RhoA-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, a negative regulator of the RhoA-ROCK-MRLC pathway, enhances motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction through activation of the myosin regulatory light chain. RhoA also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia enhances motuporamine activity, suggesting that the motuporamines act on dia in a RhoA-independent manner. Reduction in gene dose of the Drosophila Rac orthologs also enhances motuporamine activity. In contrast, motuporamine activity is unaltered by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac, the enhanced activity of motuporamines observed when Rac gene dose is reduced may reflect an indirect mode of action on the Rac GTPases leading to increased RhoA activity. In summary, these findings demonstrate that motuporamines act through RhoA and diaphanous to regulate actin-myosin contractility and actin polymerization. Citation Format: Laurence von Kalm, Corey Seavey. The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2461.

Arachidonic Acid as Mechanotransducer of Renin Cell Baroreceptor.

For normal maintenance of blood pressure and blood volume a well-balanced renin-angiotensin-aldosterone system (RAS) is necessary. For this purpose, renin is secreted as the situation demands by the juxtaglomerular cells (also called as granular cells) that are in the walls of the afferent arterioles. Juxtaglomerular cells can sense minute changes in the blood pressure and blood volume and accordingly synthesize, store, and secrete appropriate amounts of renin. Thus, when the blood pressure and blood volume are decreased JGA cells synthesize and secrete higher amounts of renin and when the blood pressure and blood volume is increased the synthesis and secretion of renin is decreased such that homeostasis is restored. To decipher this important function, JGA cells (renin cells) need to sense and transmit the extracellular physical forces to their chromatin to control renin gene expression for appropriate renin synthesis. The changes in perfusion pressure are sensed by Integrin β1 that is transmitted to the renin cell’s nucleus via lamin A/C that produces changes in the architecture of the chromatin. This results in an alteration (either increase or decrease) in renin gene expression. Cell membrane is situated in an unique location since all stimuli need to be transmitted to the cell nucleus and messages from the DNA to the cell external environment can be conveyed only through it. This implies that cell membrane structure and integrity is essential for all cellular functions. Cell membrane is composed to proteins and lipids. The lipid components of the cell membrane regulate its (cell membrane) fluidity and the way the messages are transmitted between the cell and its environment. Of all the lipids present in the membrane, arachidonic acid (AA) forms an important constituent. In response to pressure and other stimuli, cellular and nuclear shape changes occur that render nucleus to act as an elastic mechanotransducer that produces not only changes in cell shape but also in its dynamic behavior. Cell shape changes in response to external pressure(s) result(s) in the activation of cPLA2 (cytosolic phospholipase 2)-AA pathway that stretches to recruit myosin II which produces actin-myosin cytoskeleton contractility. Released AA can undergo peroxidation and peroxidized AA binds to DNA to regulate the expression of several genes. Alterations in the perfusion pressure in the afferent arterioles produces parallel changes in the renin cell membrane leading to changes in renin release. AA and its metabolic products regulate not only the release of renin but also changes in the vanilloid type 1 (TRPV1) expression in renal sensory nerves. Thus, AA and its metabolites function as intermediate/mediator molecules in transducing changes in perfusion and mechanical pressures that involves nuclear mechanotransduction mechanism. This mechanotransducer function of AA has relevance to the synthesis and release of insulin, neurotransmitters, and other soluble mediators release by specialized and non-specialized cells. Thus, AA plays a critical role in diseases such as diabetes mellitus, hypertension, atherosclerosis, coronary heart disease, sepsis, lupus, rheumatoid arthritis, and cancer.

Abstract P221: Discovery of a covalent inhibitor for an oncogenic mutant RhoAY42C

Abstract Ras homolog family member A (RhoA) is a member of a larger family of Rho proteins of small GTPases, with structural and signaling similarities to Ras proteins. RhoA is involved in multiple cellular processes including cytoskeleton regulation and actin-myosin contractility, etc. Recent studies have shown that the most common RhoA mutation in diffuse gastric cancer (DGC), Y42C, causes a gain-of-function and is associated with the loss of the E-cadherin gene CDH (CDH1). Inhibitors of RhoAY42C could be promising agents for treating DGC that harbors CDH1 mutation/deletion. BridGene expressed RhoAY42C in cells, screened our proprietary covalent library against the RhoAY42C live cells, and employed our chemoproteomic platform IMTACTM (Isobaric Mass Tagged Affinity Characterization) to detect covalent labeling of the mutant RhoA protein. A pilot screening led to the successful identification of a covalent ligand of RhoAY42C, BGS1933. Mass spec analysis has confirmed the covalent modification of Cys42 in RhoA by BGS1933. Dose-dependent binding of BGS1933 to RhoAY42C was measured using an orthogonal fluorescence-based assay. The Cancer Genome Atlas (TCGA) study shows that ~1% DGC patients harbor the oncogenic mutant RhoAY42C. The discovery of a covalent inhibitor of RhoAY42C will enable us to validate the role of RhoAY42C in DGC and facilitate the development of new first-in-class targeted therapies for the treatment of gastric cancer. Citation Format: Shirley Guo, Ping Cao. Discovery of a covalent inhibitor for an oncogenic mutant RhoAY42C [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P221.

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells.

Downregulation of Cytoskeletal Protein Kazrin in Lung Endothelial Cells of Preclinical Sickle Cell Disease Mouse Model - Essential Role of Endothelial Barrier Function

Background: Lung capillary barrier dysfunction is common to all causes of acute chest syndrome (ACS), a leading cause of morbidity and mortality in individuals with sickle cell disease (SCD). Our published data (Haematologica 2017, 102, e26-e29) demonstrated, for the first time, an impaired barrier function in freshly isolated endothelial cells (EC) from the lungs of Townes transgenic sickle cell anemia (SS) mice, compared to heterozygote (AS) or control (AA) mice. In addition, we showed a sub-stimulatory dose (0.05 mg/kg) of bacterial toxin lipopolysaccharide, which did not cause prominent acute lung injury in control AS/AA mice, was fatal to SS mice suggesting an enhanced hypersensitivity of sickle EC to infection. Hemolysis is a fundamental feature of SCD that contributes to its pathophysiology. Hemolysis results in the leakage of vast amounts of hemoglobin, and subsequently, heme into the plasma. Although, previous studies have demonstrated the detrimental effect of heme in SCD, the mechanisms of heme-mediated lung EC barrier dysfunction have not been studied. Recently, Kazrin was identified as a widely expressed protein influencing intercellular adhesion and co-localizes with the cortical actin ring to stabilize cell-cell junctions. In this study, we investigated the mechanisms of heme-induced EC barrier dysfunction related to Kazrin function, using primary EC isolated from the lungs of SCD mice and controls. Methods: Primary EC from the lungs of SS, AS, and AA mice (6-8 weeks old) were isolated according to methods described in our recent publication. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) and Evans blue dye transendothelial permeability was done using a transwell filter assay system. Western blot was performed to measure total and phosphorylated expression levels of protein involved in the regulation of EC barrier function. Immunocytochemistry was performed using specific antibodies and actin cytoskeleton was identified using a known F-actin binding fluorescent marker Phalloidin. Results: Our data demonstrated a significantly reduced basal expression of the Kazrin mRNA and protein in freshly isolated SS-EC compared to AS-EC or AA-EC. To determine the effects of Kazrin downregulation on the EC barrier function, we depleted the Kazrin protein in AS-EC using Kazrin specific siRNA and scrambled control siRNA. Kazrin depleted cells demonstrate an attenuated EC barrier function as evidenced by reduced trans-endothelial electrical resistance by ECIS and increased Evans blue dye leak by transwell filter assay (p<0.05). By contrast, control siRNA treated AS-EC displayed normal barrier function. Therefore, our data, for the first time, suggest that Kazrin is essential for the EC barrier function. Given the crucial role of hemolysis in SCD, we detected that clinically relevant dose of heme (5 µM; 60 min) significantly decreases the AS-EC barrier function (p<0.05) and the heme-mediated decrease of barrier function is dose-dependent. Moreover prolonged treatment of AS-EC with heme (5 µM; 24-48 hrs) downregulated the Kazrin protein levels. These results are intriguing since the SS-EC inherently show downregulated Kazrin when compared with AS-EC. Therefore, it is possible that impaired barrier function in SS-EC that we recently published could be an effect of Kazrin downregulation since hemolysis is a constant process in SCD. We also found that incubation of AS-EC with heme upregulated the efficacy of barrier disruption agent RhoA. Previous studies have indicated that activated RhoA promotes the actin stress fibers by stimulating actin-myosin contractility and increased endothelial permeability. Our immunocytochemistry data reveal that heme incubated AS-EC develop severe actin stress fibers. Studies in progress to determine the mechanistic link between Kazrin downregulation, RhoA activation and impaired EC barrier function in SS-EC. Conclusions and Future Direction: Our findings support the notion that Kazrin participates in the EC barrier function and its downregulation cause an impaired EC barrier function. Our future studies will focus on to identify protective mechanisms of lung EC barrier function in SCD. Disclosures Kutlar: Bluebird Bio: Other: DSMB Member; Novo Nordisk: Research Funding; Novartis: Consultancy; Micelle Biopharma: Other: DSMB Chair; Global Blood Therapeutics, Inc. (GBT): Research Funding.

Actin dynamics and myosin contractility during plasma membrane repair and restoration: Does one ring really heal them all?

In order to survive daily insults, cells have evolved various mechanisms that detect, stabilize and repair damages done to their plasma membrane and cytoskeletal structures. Damage to the PM endangers wounded cells by exposing them to uncontrolled exchanges with the extracellular milieu. The processes and molecular machinery enabling PM repair are therefore at the center of the bulk of the investigations into single-cell repair program. Wounds are repaired by dynamically remodeling the composition and shape of the injured area through exocytosis-mediated release of intracellular membrane components to the wounded area, endocytosis-mediated removal of the injured area, or the shedding of the injury. The wound healing program of Xenopus oocytes and early Drosophila embryos is by contrast, mostly characterized by the rapid formation of a large membrane patch over the wound that eventually fuse with the plasma membrane which restores plasma membrane continuity and lead to the shedding of patch material into the extracellular space. Formation and contraction of actomyosin ring restores normal plasma membrane composition and organizes cytoskeletal repairs. The extend of the contributions of the cytoskeleton to the wound healing program of somatic cells have comparatively received little attention. This review offers a survey of the current knowledge on how actin dynamics, myosin-based contraction and other cytoskeletal structures affects PM and cortical cytoskeleton repair of somatic cells.

Variations on the theme of podosomes, context matters

Podosomes are actin-based microdomains connecting the cell with its extracellular matrix. Contractile actin-myosin cables assemble them into a network that constitutes a versatile cellular superstructure. Discovered and extensively described in in vitro conditions, podosomes now appear as major actors of specific physiological processes. They share common characteristics but their morphology and their effect on cell functioning can only be apprehended in specific cellular contexts. We focus here on three cellular processes involving podosomes and discuss their properties in context.

Abstract 1933: A novel small molecule inhibitor of MRCK shows utility in blocking radiation induced invasion of glioblastoma cells in vitro and in vivo

Abstract Myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and are members of the AGC family of serine/threonine kinases. MRCKs are closely related to the Rho-regulated ROCK kinases, which is reflected in their shared abilities to phosphorylate a similar set of substrates, including myosin II light chain proteins (MLC) and myosin phosphatase target subunit 1 (MYPT1). However, MRCK and ROCK may phosphorylate these substrates at different subcellular localisations leading to diverging effects on cell invasion and migration. Glioblastoma (GBM) is an aggressive and incurable primary brain tumor. Patients are treated with surgery, radiotherapy and chemotherapy but prognosis remains poor, with a median survival of 15 months. Complete surgical resection of GBM is difficult because these tumours are frequently invasive. Recent research has indicated that, while radiotherapy extends life expectancy of patients, it can provoke a more infiltrative phenotype in those GBM cells that survive treatment. We have developed potent and selective small molecule MRCK inhibitors exemplified by BDP-9066, which inhibits pMLC in a selective cellular assay with an EC50 28.3 nM, whilst displaying high selectivity over ROCK1 where the EC50 10.6 µM. This compound, and others in the series, have been used in numerous cellular assay systems to demonstrate that MRCK inhibition suppresses the invasion and migration of cancer cells including, breast, skin SCC and glioblastoma. We have used BDP-9066 to demonstrate that MRCK inhibition is effective at perturbing radiation-induced migration of GBM cells in culture at concentrations that block phosphorylation of MLC. Using a clinically relevant intracranial mouse model that recapitulates key histological features of the disease, we have shown that BDP-9066 inhibits the invasion of G7 GBM cells into the contralateral hemisphere of the brain. These experiments highlight the potential utility of MRCK inhibition in the treatment of GBM in patients. Citation Format: Heather J. McKinnon, Joanna Birch, Laura McDonald, Mairi Sime, Daniel Croft, Diane Crighton, Martin Drysdale, Lesley Gilmore, Christopher Gray, Jennifer Konczal, Duncan McArthur, Patricia McConnell, Mokdad Mezna, Alexander Schuettelkopf, Karen Strathdee, Justin Bower, Michael F. Olson, Anthony J. Chalmers. A novel small molecule inhibitor of MRCK shows utility in blocking radiation induced invasion of glioblastoma cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1933.

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Cytotoxic Tcells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.

A free-boundary model of a motile cell explains turning behavior.

To understand shapes and movements of cells undergoing lamellipodial motility, we systematically explore minimal free-boundary models of actin-myosin contractility consisting of the force-balance and myosin transport equations. The models account for isotropic contraction proportional to myosin density, viscous stresses in the actin network, and constant-strength viscous-like adhesion. The contraction generates a spatially graded centripetal actin flow, which in turn reinforces the contraction via myosin redistribution and causes retraction of the lamellipodial boundary. Actin protrusion at the boundary counters the retraction, and the balance of the protrusion and retraction shapes the lamellipodium. The model analysis shows that initiation of motility critically depends on three dimensionless parameter combinations, which represent myosin-dependent contractility, a characteristic viscosity-adhesion length, and a rate of actin protrusion. When the contractility is sufficiently strong, cells break symmetry and move steadily along either straight or circular trajectories, and the motile behavior is sensitive to conditions at the cell boundary. Scanning of a model parameter space shows that the contractile mechanism of motility supports robust cell turning in conditions where short viscosity-adhesion lengths and fast protrusion cause an accumulation of myosin in a small region at the cell rear, destabilizing the axial symmetry of a moving cell.

Tanshinone I prevents atorvastatin-induced cerebral hemorrhage in zebrafish and stabilizes endothelial cell–cell adhesion by inhibiting VE-cadherin internalization and actin-myosin contractility

Defects in vascular integrity in cerebrovasculature lead to serious pathologies including hemorrhagic stroke. The stability of cell adhesion junctions and actin-myosin contractile machinery are two major determinants for the integrity of endothelial monolayer. Here we have evaluated the protective effects of tanshinone I (Tan I), a lipophilic compound presents in Salvia miltiorrhiza, against atorvastatin-induced cerebral hemorrhage in zebrafish in vivo, and further dissected the molecular mechanisms in HUVECs. We demonstrated that Tan I protected endothelial integrity by stabilizing cell–cell adhesion junctions via the inhibition of Src-mediated VE-cadherin internalization and subsequent junction-linked actin cytoskeleton depolymerization. In addition, Tan I inhibited ROCK-associated endothelial contractile machinery by dephosphorylating cofilin and MYPT1. These findings identified Tan I as an endothelial stabilizing agent and suggested Tan I as a potential treatment for vascular leakage in hemorrhagic stroke.

The shift in GH3 cell shape and cell motility is dependent on MLCK and ROCK

Cytoskeletal organization, actin-myosin contractility and the cell membrane together regulate cell morphology in response to the cell environment, wherein the extracellular matrix (ECM) is an indispensable component. Plasticity in cell shape enables cells to adapt their migration mode to their surroundings. GH3 endocrine cells respond to different ECM proteins, acquiring different morphologies: a rounded on collagen I-III (C I-III) and an elongated on collagen IV (C IV). However, the identities of the molecules that participate in these responses remain unknown. Considering that actin-myosin contractility is crucial to maintaining cell shape, we analyzed the participation of MLCK and ROCK in the acquisition of cell shape, the generation of cellular tension and the cell motility mode. We found that a rounded shape with high cortical tension depends on MLCK and ROCK, whereas in cells with an elongated shape, MLCK is the primary protein responsible for cell spreading. Further, in cells with a slow and directionally persistent motility, MLCK predominates, while rapid and erratic movement is ROCK-dependent. This behavior also correlates with GTPase activation. Cells on C I-III exhibited higher Rho-GTPase activity than cells on C IV and vice versa with Rac-GTPase activity, showing a plastic response of GH3 cells to their environment, leading to the generation of different cytoskeleton and membrane organizations and resulting in two movement strategies, rounded and fibroblastoid-like.

Force Generation by T Cells Modulates the Strength of Activation

T cell activation is a key step in the adaptive immune response and involves formation of a complex signaling structure by both the T cell and antigen presenting cell (APC). The process of activation is punctuated by dramatic reorganization of the T cell's cytoskeleton at the synapse. In order to measure forces generated by T cells at the T-APC interface and understand how they modulate activation strength, we developed a novel application of atomic force microscopy (AFM). We used chemically modified AFM cantilevers to present antigenic signal to primary T cells while imaging optically and measuring forces exerted by the cell on the cantilever. T cells exert significant push and pull forces during activation, mediated by actin network growth and myosin contractility. Inhibition of actin polymerization greatly weakened force generation and T cell activation. We were able to emulate T cell generated forces using movement of the cantilever, partially rescuing activation in these actin-inhibited T cells. Furthermore, negation of cell-generated forces by “force-clamping” the cantilever resulted in decreased T cell sensitivity to antigenic signals. Our studies suggest a feedback loop wherein initial TCR engagement leads to force generation by the T cell against the APC to enhance signaling strength allowing increased sensitivity to cognate antigen.

Autonomous osteogenic differentiation of hASCs encapsulated in methacrylated gellan-gum hydrogels

Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels – excluding eventual on-chip crosstalk – and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs’ osteodifferentiation. GG-MA hydrogels also did not promote hASCs’ adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial. Statement of SignificanceMethacrylated gellan gum (GG-MA) is here suggested for the first time as a widely available polysaccharide to easily prepare hydrogels with cell adhesion properties and capability of inducing the autonomous osteogenic differentiation of human adipose-derived stem cells (hASCs). GG-MA was processed as stand-alone hydrogels or in different combinations with collage type I. All hydrogel formulations elicited the osteogenic differentiation of hASCs, independently of the addition of any osteoconductive or osteogenic stimuli, i.e. in basal/growth medium. Effective cellular adhesion to methacrylated gellan gum hydrogels in the absence of any cell-ligand peptide/protein was here proved for the first time. Moreover, we showed that the encapsulated hASCs underwent osteogenic differentiation due to a mechanotransduction phenomenon dependent on the actin-myosin contractility pathway.

Rho Kinase Inhibition Is Essential During In Vitro Neurogenesis and Promotes Phenotypic Rescue of Human Induced Pluripotent Stem Cell-Derived Neurons With Oligophrenin-1 Loss of Function.

: Rho-GTPases have relevant functions in various aspects of neuronal development, such as differentiation, migration, and synaptogenesis. Loss of function of the oligophrenin-1 gene (OPHN1) causes X-linked intellectual disability with cerebellar hypoplasia and leads to hyperactivation of the rho kinase (ROCK) pathway. ROCK mainly acts through phosphorylation of the myosin phosphatase targeting subunit 1, triggering actin-myosin contractility. We show that during in vitro neurogenesis, ROCK activity decreases from day 10 until terminal differentiation, whereas in OPHN1-deficient human induced pluripotent stem cells (h-iPSCs), the levels of ROCK are elevated throughout differentiation. ROCK inhibition favors neuronal-like appearance of h-iPSCs, in parallel with transcriptional upregulation of nuclear receptor NR4A1, which is known to induce neurite outgrowth. This study analyzed the morphological, biochemical, and functional features of OPHN1-deficient h-iPSCs and their rescue by treatment with the ROCK inhibitor fasudil, shedding light on the relevance of the ROCK pathway during neuronal differentiation and providing a neuronal model for human OPHN1 syndrome and its treatment. The analysis of the levels of rho kinase (ROCK) activity at different stages of in vitro neurogenesis of human induced pluripotent stem cells reveals that ROCK activity decreases progressively in parallel with the appearance of neuronal-like morphology and upregulation of nuclear receptor NR4A1. These results shed light on the role of the ROCK pathway during early stages of human neurogenesis and provide a neuronal stem cell-based model for the treatment of OPHN1 syndrome and other neurological disorders due to ROCK dysfunction.

From Nanoscale to Mesoscale: Integrating Advanced Microscopy Techniques to Reveal the Ultrastructure and Coordinated Dynamics of Mechanosensory Podosomes

Podosomes are dynamic integrin- and actomyosin-based multi-molecular adhesion structures involved in cell protrusion, topography sensing and extracellular matrix degradation. Formed by a variety of cells, they have a dotted shape and undergo irregular cycles of growth and shrinkage driven by actin polymerization and myosin contractility. With an actine core of ∼0.5um, hundreds of podosomes organize in different mesoscale spatial arrangements ranging from large circular belts in bone-degrading osteoclasts to small rosettes in endothelial cells and well-defined large clusters in antigen-presenting cells. The molecular mechanisms regulating formation and maintenance of these mesoscale arrangements are still poorly defined. By integrating a variety of advanced microscopy techniques, we aim to unravel the nanoscale structural and dynamic complexity of individual podosomes as well as formation, architecture and function of mesoscale podosome clusters. We previously demonstrated that, at the nanoscale, single podosomes comprise a dense actin core with radiating actin filaments decorated by vinculin, and that integrins and talin densely cover the entire cluster area except the actin core regions. At present, by exploiting an extension of spatiotemporal image correlation spectroscopy (STICS), we measured the evolution of spatiotemporal molecular dynamics of podosome components, revealing self-organized dynamic spatial patterns of various cytoskeletal components throughout the clusters. Furthermore, by multi-color SIM and 3D STORM, we revealed the presence of actin substructures regulating the core nanoscale architecture that likely contribute to their protrusive behaviour and tensional integrity. Together, our findings propose podosomes as highly organized actin microarchitectures mediating tensional integrity at the nanoscale and providing a feedback mechanism for cellular mechanosensing at the mesoscale.

A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

BackgroundThe myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.ResultsScreening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 μM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.ConclusionsBDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

Cdh5/VE-cadherin Promotes Endothelial Cell Interface Elongation via Cortical Actin Polymerization during Angiogenic Sprouting

Organ morphogenesis requires the coordination of cell behaviors. Here, we have analyzed dynamic endothelial cell behaviors underlying sprouting angiogenesis invivo. Two different mechanisms contribute to sprout outgrowth: tip cells show strong migratory behavior, whereas extension of the stalk isdependent upon cell elongation. To investigate the function of Cdh5 in sprout outgrowth, we generated null mutations in the zebrafish cdh5 gene, and we found that junctional remodeling and cell elongation are impaired in mutant embryos. The defects are associated with a disorganization of the actin cytoskeleton and cannot be rescued by expression of atruncated version of Cdh5. Finally, the defects in junctional remodeling can be phenocopied by pharmacological inhibition of actin polymerization, but not by inhibiting actin-myosin contractility. Taken together, our results support a model in which Cdh5 organizes junctional and cortical actin cytoskeletons, as well as provides structural support for polymerizing F-actin cables during endothelial cell elongation.