Regulation of global CD8+ T-cell positioning by the actomyosin cytoskeleton.

CD8+ T cells have evolved as one of the most motile mammalian cell types, designed to continuously scan peptide-major histocompatibility complexes class I on the surfaces of other cells. Chemoattractants and adhesion molecules direct CD8+ T-cell homing to and migration within secondary lymphoid organs, where these cells colocalize with antigen-presenting dendritic cells in confined tissue volumes. CD8+ T-cell activation induces a switch to infiltration of non-lymphoid tissue (NLT), which differ in their topology and biophysical properties from lymphoid tissue. Here, we provide a short overview on regulation of organism-wide trafficking patterns during naive T-cell recirculation and their switch to non-lymphoid tissue homing during activation. The migratory lifestyle of CD8+ T cells is regulated by their actomyosin cytoskeleton, which translates chemical signals from surface receptors into mechanical work. We explore how properties of the actomyosin cytoskeleton and its regulators affect CD8+ T cell function in lymphoid and non-lymphoid tissue, combining recent findings in the field of cell migration and actin network regulation with tissue anatomy. Finally, we hypothesize that under certain conditions, intrinsic regulation of actomyosin dynamics may render NLT CD8+ T-cell populations less dependent on input from extrinsic signals during tissue scanning.

HAND2-mediated epithelial maintenance and integrity in cardiac outflow tract morphogenesis.

During embryogenesis, epithelial organization is the prerequisite for organogenesis, in particular, for establishing the tubular structure. Recent studies provided hints about epithelial formation in early heart development, which has not been systemically explored. Here, we revealed a gradient of HAND2 protein in cardiac progenitors in the anterior dorsal pericardial wall (aDPW) and adjacent transition zone (TZ) in the outflow tract (OFT). Deletion of Hand2 caused cell arrest and accumulation in the TZ, leading to defective morphogenesis. Although apicobasal cell polarity was unaffected, the key epithelial elements of adherens junction and cell-matrix adhesion were disrupted in the TZ of Hand2 mutant mice, indicating poorly formed epithelium. RNA-seq analysis revealed altered regulation of the contractile fiber and actin cytoskeleton, which affected cardiomyocyte differentiation. Furthermore, we have identified Stars as being transcriptionally controlled by HAND2. STARS facilitates actin polymerization that is essential for anchoring the adhesive molecules to create cell adhesion. Thus, we have uncovered a new function of HAND2 in mediating epithelial maintenance and integrity in OFT morphogenesis. In addition, this study also provides insights for understanding cardiac progenitor contribution to OFT development.

Death for life: a path from apoptotic signaling to tissue-scale effects of apoptotic epithelial extrusion.

Apoptosis plays a crucial role in clearing old or critically compromised cells, and actively maintains epithelial homeostasis and epithelial morphogenesis during embryo development. But how is the apoptotic signaling pathway able to orchestrate such complex and dynamic multi-cellular morphological events at the tissue scale? In this review we collected the most updated knowledge regarding how apoptosis controls different cytoskeletal components. We describe how apoptosis can control epithelial homeostasis though epithelial extrusion, a highly orchestrated process based on high- order actomyosin structures and on the coordination between the apoptotic and the neighboring cells. Finally, we describe how the synergy among forces generated by multiple apoptotic cells can shape epithelia in embryo development.

Rho-ROCK Signaling in Normal Physiology and as a Key Player in Shaping the Tumor Microenvironment.

The Rho-ROCK signaling network has a range of specialized functions of key biological importance, including control of essential developmental processes such as morphogenesis and physiological processes including homeostasis, immunity, and wound healing. Deregulation of Rho-ROCK signaling actively contributes to multiple pathological conditions, and plays a major role in cancer development and progression. This dynamic network is critical in modulating the intricate communication between tumor cells, surrounding diverse stromal cells and the matrix, shaping the ever-changing microenvironment of aggressive tumors. In this chapter, we overview the complex regulation of the Rho-ROCK signaling axis, its role in health and disease, and analyze progress made with key approaches targeting the Rho-ROCK pathway for therapeutic benefit. Finally, we conclude by outlining likely future trends and key questions in the field of Rho-ROCK research, in particular surrounding Rho-ROCK signaling within the tumor microenvironment.

Src-Dependent NM2A Tyrosine Phosphorylation Regulates Actomyosin Remodeling.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr158) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr158 defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr158 as a novel layer of regulation of actomyosin cytoskeleton organization.

Building the synapse engine to drive B lymphocyte function.

B cell receptor (BCR)-mediated antigen-specific recognition activates B lymphocytes and drives the humoral immune response. This enables the generation of antibody-producing plasma cells, the effector arm of the B cell immune response, and of memory B cells, which confer protection against additional encounters with antigen. B cells search for cognate antigen in the complex cellular microarchitecture of secondary lymphoid organs, where antigens are captured and exposed on the surface of different immune cells. While scanning the cell network, the BCR can be stimulated by a specific antigen and elicit the establishment of the immune synapse with the antigen-presenting cell. At the immune synapse, an integrin-enriched supramolecular domain is assembled at the periphery of the B cell contact with the antigen-presenting cell, ensuring a stable and long-lasting interaction. The coordinated action of the actomyosin cytoskeleton and the microtubule network in the inner B cell space provides a structural framework that integrates signaling events and antigen uptake through the generation of traction forces and organelle polarization. Accordingly, the B cell immune synapse can be envisioned as a temporal engine that drives the molecular mechanisms needed for successful B cell activation. Here, I review different aspects of the B cell synapse engine and provide insights into other aspects poorly known or virtually unexplored.

HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis.

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCßII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCßII, by coactivating Sp1, and triggered PKCßII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCßII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Integrating mechanical signals into cellular identity.

The large arrays of cell types in a multicellular organism are defined by their stereotypic size and/or morphology, and, for cells in vivo, by their anatomic positions. Historically, this identity-structure-function correlation was conceptualized as arising from distinct gene expression programs that dictate how cells appear and behave. However, a growing number of studies suggest that a cell's mechanical state is also an important determinant of its identity, both in lineage-committed cells and in pluripotent stem cells. Defining the mechanism by which mechanical inputs influence complex cellular programs remains an area of ongoing investigation. Here, we discuss how the cytoskeleton actively participates in instructing the response of the nucleus and genome to integrate mechanical and biochemical inputs, with a primary focus on the role of the actomyosin-LINC (linker of nucleoskeleton and cytoskeleton) complex axis.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways.

The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells.

OBJECTIVES: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells. MATERIAL AND METHODS: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues. RESULTS: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin. CONCLUSIONS: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.

Quantitative Analysis of Myofibroblast Contraction by Traction Force Microscopy.

Myofibroblasts play important roles in physiological processes such as wound healing and tissue repair. While high contractile forces generated by the actomyosin network enable myofibroblasts to physically contract the wound and bring together injured tissue, prolonged and elevated levels of contraction also drive the progression of fibrosis and cancer. However, quantitative mapping of these forces has been difficult due to their extremely low magnitude ranging from 100 pN/µm2 to 2 nN/µm2. Here, we provide a protocol to measure cellular forces exerted on two-dimensional compliant elastic hydrogels. We describe the fabrication of polyacrylamide hydrogels labeled with fluorescent fiducial markers, functionalization of substrates with ECM proteins, setting up the experiment, and imaging procedures. We demonstrate the application of this technique for quantitative analysis of traction forces exerted by myofibroblasts.

Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defects in In Vitro Endothelial CCM Model: Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells.

Endothelial cells lining cerebral cavernous malformations (CCM) present strong adhesive and mechanical defects. Increased cell contractility is a driver to the onset and the expansion of the CCM lesions. 2D in vitro endothelial models have been developed from either endothelial cells isolated from ccm1-3 knock-out mice or CCM1-3-silenced primary endothelial cells. These in vitro models faithfully recapitulate the adhesive and contractile defects of the CCM-deficient endothelial cells such as increased cell-extracellular matrix (ECM) adhesion through ß1 integrin-anchored actin stress fibers, abnormal remodeling of the ECM, and destabilized VE-cadherin-dependent cell-cell junctions. Using such 2D in vitro CCM models, we have shown that the ECM remodeled by CCM-depleted endothelial cells can propagate CCM-like adhesive defects to wild-type endothelial cells, a process potentially pertinent to CCM lesion expansion. Here, we detail methods for studying the morphology of focal adhesions, actomyosin cytoskeleton, and VE-cadherin-dependent Adherens junctions by immunofluorescence and morphometric analyses. Moreover, we detail the protocols to produce and purify remodeled ECM and to test its effect on endothelial cell adhesion.

Non-Muscle Myosin II in Axonal Cell Biology: From the Growth Cone to the Axon Initial Segment.

By binding to actin filaments, non-muscle myosin II (NMII) generates actomyosin networks that hold unique contractile properties. Their dynamic nature is essential for neuronal biology including the establishment of polarity, growth cone formation and motility, axon growth during development (and axon regeneration in the adult), radial and longitudinal axonal tension, and synapse formation and function. In this review, we discuss the current knowledge on the spatial distribution and function of the actomyosin cytoskeleton in different axonal compartments. We highlight some of the apparent contradictions and open questions in the field, including the role of NMII in the regulation of axon growth and regeneration, the possibility that NMII structural arrangement along the axon shaft may control both radial and longitudinal contractility, and the mechanism and functional purpose underlying NMII enrichment in the axon initial segment. With the advances in live cell imaging and super resolution microscopy, it is expected that in the near future the spatial distribution of NMII in the axon, and the mechanisms by which it participates in axonal biology will be further untangled.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin.

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Non-Muscle Myosin 2A (NM2A): Structure, Regulation and Function.

Non-muscle myosin 2A (NM2A) is a motor cytoskeletal enzyme with crucial importance from the early stages of development until adulthood. Due to its capacity to convert chemical energy into force, NM2A powers the contraction of the actomyosin cytoskeleton, required for proper cell division, adhesion and migration, among other cellular functions. Although NM2A has been extensively studied, new findings revealed that a lot remains to be discovered concerning its spatiotemporal regulation in the intracellular environment. In recent years, new functions were attributed to NM2A and its activity was associated to a plethora of illnesses, including neurological disorders and infectious diseases. Here, we provide a concise overview on the current knowledge regarding the structure, the function and the regulation of NM2A. In addition, we recapitulate NM2A-associated diseases and discuss its potential as a therapeutic target.

Quantitative Analysis Reveals that Actin and Src-Family Kinases Regulate Nuclear YAP1 and Its Export.

The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus.

HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling.

During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. Here, we show that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. We reveal that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.