Renal fibrosis, a major risk factor for kidney failure, can lead to chronic kidney disease (CKD) and is caused by cytoskeleton reorganization and mitochondrial dysfunction. In this study, we ...investigated the potential of melatonin treatment to reduce renal fibrosis by recovering the cytoskeleton reorganization and mitochondrial dysfunction. We found that miR-4516 expression was downregulated in the renal cortex of CKD mice and
-cresol-treated TH1 cells. Decreased miR-4516 expression stimulated cytoskeleton reorganization and mitochondrial dysfunction, and induced renal fibrosis. Melatonin treatment suppressed fibrosis by inhibiting cytoskeleton reorganization and restoring mitochondrial function via increased miR-4516 expression. More specifically, melatonin treatment increased miR-4516 expression while decreasing ITGA9 expression, thereby inhibiting cytoskeleton reorganization. In addition, increased expression of miR-4516 by melatonin treatment reduced ROS formation and restored mitochondrial function. These findings suggest that melatonin may be a promising treatment for patients with CKD having renal fibrosis. Moreover, regulation of miR-4516 expression may be a novel strategy for the treatment of renal fibrosis.
Glioblastoma (GBM) is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy. GBM tumors show nuclear factor-κB (NF-κB) activity that has been associated ...with tumor formation, growth, and increased resistance to therapy. We investigated the effect of NF-κB inhibitor BAY 11-7082 with Temozolomide (TMZ) on the signaling pathways in GBM pathogenesis. GBM cells and patient-derived GBM cells cultured in 3D microwells were co-treated with BAY 11-7082 and TMZ or BAY 11-7082 and TMZ alone, and combined experiments of cell proliferation, apoptosis, wound healing assay, as well as reverse-phase protein arrays, western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells. The results revealed that the co-treatment significantly altered cell proliferation by decreasing GBM viability, suppressed NF-κB pathway and enhanced apoptosis. Moreover, it was found that the co-treatment of BAY 11-7082 and TMZ significantly contributed to a decrease in the migration pattern of patient-derived GBM cells by modulating actin cytoskeleton pathway. These findings suggest that in addition to TMZ treatment, NF-κB can be used as a potential target to increase the treatment's outcomes. The drug combination strategy, which is significantly improved by NF-κB inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatment.
Abstract Cells sense their physical microenvironment and transduce these signals through actin-nuclear links to regulate nuclear functions including gene expression. However, the spatio-temporal ...coupling between perinuclear actin and nucleus and their functional importance are still unclear. Using micropatterned substrates to control cell geometry, we show that perinuclear actin organization at the apical plane remodels from mesh-like structure to stress fibers. The formation of these apical stress fibers (ASFs) correlated with significant reduction in nuclear height and was found to exert an active compressive load on the nucleus via direct contact with mature focal adhesion sites. Interestingly, the dynamic nature of ASFs was found to transduce forces to chromatin assembly. In addition, geometric perturbations or using pharmacological drugs to inhibit actomyosin contractility of ASFs resulted in nuclear instability. Taken together, our work provides direct evidence of physical links between the nucleus and focal adhesion sites via ASFs, which modulate nuclear homeostatic balance and internal chromatin structure. We suggest that such direct links may underlie nuclear mechanotransduction to regulate genomic programs.
Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin‐associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, ...intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.
Mechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in ...cytoskeletal proteins and transcriptional regulators. Here, we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding as a mechanosensing mechanism by which cytoskeletal tension can govern nuclear localization.
Display omitted
•Tension recruits many tandem LIM domain proteins to the actin cytoskeleton•A conserved phenylalanine in each LIM domain is required for this localization•Mechanoresponsive LIM proteins directly bind F-actin only in the presence of force•This binding governs the ECM-rigidity-dependent nuclear localization of FHL2
Sun et al. show that mechanoresponsive tandem LIM domain proteins directly bind F-actin only in the presence of force through a conserved mechanism, governing their tension-dependent cytoskeletal localization in cells. Binding tensed cytoplasmic F-actin prevents the transcriptional co-activator FHL2 from entering the nucleus, facilitating mechanosensing through the cytoskeleton.
Leading edge protrusion of migrating cells involves tightly coordinated changes in the plasma membrane and actin cytoskeleton. It remains unclear whether polymerizing actin filaments push and deform ...the membrane, or membrane deformation occurs independently and is subsequently stabilized by actin filaments. To address this question, we employed an ability of the membrane-binding I-BAR domain of IRSp53 to uncouple the membrane and actin dynamics and to induce filopodia in expressing cells. Using time-lapse imaging and electron microscopy of IRSp53-I-BAR-expressing B16F1 melanoma cells, we demonstrate that cells are not able to protrude or maintain durable long extensions without actin filaments in their interior, but I-BAR-dependent membrane deformation can create a small and transient space at filopodial tips that is subsequently filled with actin filaments. Moreover, the expressed I-BAR domain forms a submembranous coat that may structurally support these transient actin-free protrusions until they are further stabilized by the actin cytoskeleton. Actin filaments in the I-BAR-induced filopodia, in contrast to normal filopodia, do not have a uniform length, are less abundant, poorly bundled, and display erratic dynamics. Such unconventional structural organization and dynamics of actin in I-BAR-induced filopodia suggests that a typical bundle of parallel actin filaments is not necessary for generation and mechanical support of the highly asymmetric filopodial geometry. Together, our data suggest that actin filaments may not directly drive the protrusion, but only stabilize the space generated by the membrane deformation; yet, such stabilization is necessary for efficient protrusion.
In many organisms, cell division is driven by the constriction of a cytokinetic ring, which consists of actin filaments and crosslinking proteins. While it has long been believed that the ...constriction is driven by motor proteins, it has recently been discovered that passive crosslinkers that do not turn over fuel are able to generate enough force to constrict actin filament rings. To study the ring constriction dynamics, we develop a model that includes the driving force of crosslinker condensation and the opposing forces of friction and filament bending. We analyze the constriction force as a function of ring topology and crosslinker concentration, and predict forces that are sufficient to constrict an unadorned plasma membrane. Our model also predicts that actin-filament sliding arises from an interplay between filament rotation and crosslinker hopping, producing frictional forces that are low compared with those of crosslinker-mediated microtubule sliding.
Full text
Available for:
CMK, CTK, FMFMET, NUK, UL
How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm ...is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.
•In vitro recons,tu,on of the lamellipodia- to filopodia-like network transion.•Biomime,c recons,tu,on assays: micron-sized beads and micropatterning.•Formin and VASP are necessary and sufficient to drive network transion.•Compe, on with capping protein regulates filopodia-like network characteriscs.
The actin cortex is a complex cytoskeletal machinery that drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as ...cell division, migration, and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches associated with negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modeling in terms of membrane hemifusion of nested GUVs. We find that branched actin networks preferentially assemble at the neck of the dumbbells, which possess a micrometer-range convex curvature comparable with the curvature of the actin patches found in spherical GUVs. Minimal branched actin networks can thus sense membrane curvature, which may help mammalian cells to robustly recruit actin to curved membranes to facilitate diverse cellular functions such as cytokinesis and migration.
Cellular protrusions are typically considered as distinct structures associated with specific regulators. However, we found that these regulators coordinately localize as propagating cortical waves, ...suggesting a common underlying mechanism. These molecular events fell into two excitable networks, the signal transduction network STEN and the cytoskeletal network CEN with different wave substructures. Computational studies using a coupled‐network model reproduced these features and showed that the morphology and kinetics of the waves depended on strengths of feedback loops. Chemically induced dimerization at multiple nodes produced distinct, coordinated alterations in patterns of other network components. Taken together, these studies indicate: STEN positive feedback is mediated by mutual inhibition between Ras/Rap and PIP2, while negative feedback depends on delayed PKB activation; PKBs link STEN to CEN; CEN includes positive feedback between Rac and F‐actin, and exerts fast positive and slow negative feedbacks to STEN. The alterations produced protrusions resembling filopodia, ruffles, pseudopodia, or lamellipodia, suggesting that these structures arise from a common regulatory mechanism and that the overall state of the STEN‐CEN system determines cellular morphology.
Synopsis
Theoretical and experimental analyses indicate that signal transduction (STEN) and cytoskeletal (CEN) excitable networks interact to control dynamic wave patterns at the cell cortex. Network perturbations change wave speed and range and create distinct protrusions.
Cell cortical wave patterns are brought about by coupled Rap/Ras and Rac/F‐actin centric networks that are linked via PIP3/PKBs.
Increases in Rap/Ras or decreases in PIP2 activate while PIP3/PKBs negatively regulate STEN. Increasing Rac/F‐actin activates CEN and provides feedback to STEN.
Feedback strengths of the coupled excitable networks control speed and range of wave patterns.
Protrusive form and dynamic cell morphology are determined by the overall state of the coupled networks rather than single regulators.
Theoretical and experimental analyses indicate that signal transduction (STEN) and cytoskeletal (CEN) excitable networks interact to control dynamic wave patterns at the cell cortex. Network perturbations change wave speed and range and create distinct protrusions.
PDF
