A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin
Ontology highlight
ABSTRACT:
SUBMITTER: Quadri R
PROVIDER: S-EPMC10570125 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.
| S-EPMC2667767 | biostudies-literature
Project description:BackgroundAlzheimer's Disease (AD) is a neurodegenerative disorder characterized by the accumulation of pathological proteins and synaptic dysfunction. This study aims to investigate the molecular and functional differences between human induced pluripotent stem cells (hiPSCs) derived from patients with sporadic AD (sAD) and age-matched controls (healthy subjects, HS), focusing on their neuronal differentiation and synaptic properties in order to better understand the cellular and molecular mechanisms underlying AD pathology.MethodsSkin fibroblasts from sAD patients (n = 5) and HS subjects (n = 5) were reprogrammed into hiPSCs using non-integrating Sendai virus vectors. Through karyotyping, we assessed pluripotency markers (OCT4, SOX2, TRA-1-60) and genomic integrity. Neuronal differentiation was evaluated by immunostaining for MAP2 and NEUN. Electrophysiological properties were measured using whole-cell patch-clamp, while protein expression of Aβ, phosphorylated tau, Synapsin-1, Synaptophysin, PSD95, and GluA1 was quantified by western blot. We then focused on PAK1-LIMK1-Cofilin signaling, which plays a key role in regulating synaptic structure and function, both of which are disrupted in neurodegenerative diseases such as AD.ResultssAD and HS hiPSCs displayed similar stemness features and genomic stability. However, they differed in neuronal differentiation and function. sAD-derived neurons (sAD-hNs) displayed increased levels of AD-related proteins, including Aβ and phosphorylated tau. Electrophysiological analyses revealed that while both sAD- and HS-hNs generated action potentials, sAD-hNs exhibited decreased spontaneous synaptic activity. Significant reductions in the expression of synaptic proteins such as Synapsin-1, Synaptophysin, PSD95, and GluA1 were found in sAD-hNs, which are also characterized by reduced neurite length, indicating impaired differentiation. Notably, sAD-hNs demonstrated a marked reduction in LIMK1 phosphorylation, which could be the underlying cause for the changes in cytoskeletal dynamics that we found, leading to the morphological and functional modifications observed in sAD-hNs. To further investigate the involvement of the LIMK1 pathway in the morphological and functional changes observed in sAD neurons, we conducted perturbation experiments using the specific LIMK1 inhibitor, BMS-5. Neurons obtained from healthy subjects treated with the inhibitor showed similar morphological changes to those observed in sAD neurons, confirming that LIMK1 activity is crucial for maintaining normal neuronal structure. Furthermore, administration of the inhibitor to sAD neurons did not exacerbate the morphological alterations, suggesting that LIMK1 activity is already compromised in these cells.ConclusionOur findings demonstrate that although sAD- and HS-hiPSCs are similar in their stemness and genomic stability, sAD-hNs exhibit distinct functional and structural anomalies mirroring AD pathology. These anomalies include synaptic dysfunction, altered cytoskeletal organization, and accumulation of AD-related proteins. Our study underscores the usefulness of hiPSCs in modeling AD and provides insights into the disease's molecular underpinnings, thus highlighting potential therapeutic targets.
| S-EPMC11660640 | biostudies-literature
Project description:Neuroligin (NLG) 1 is important for synapse development and function, but the underlying mechanisms remain unclear. It is known that at least some aspects of NLG1 function are independent of the presynaptic neurexin, suggesting that the C-terminal domain (CTD) of NLG1 may be sufficient for synaptic regulation. In addition, NLG1 is subjected to activity-dependent proteolytic cleavage, generating a cytosolic CTD fragment, but the significance of this process remains unknown. In this study, we show that the CTD of NLG1 is sufficient to (a) enhance spine and synapse number, (b) modulate synaptic plasticity, and (c) exert these effects via its interaction with spine-associated Rap guanosine triphosphatase-activating protein and subsequent activation of LIM-domain protein kinase 1/cofilin-mediated actin reorganization. Our results provide a novel postsynaptic mechanism by which NLG1 regulates synapse development and function.
| S-EPMC4754719 | biostudies-literature
Project description:BackgroundEmbryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity, molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.Methodology/principal findingsHere we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock, a major effector kinase downstream of Rho, played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore, a novel culture system combining a single synthetic matrix, defined medium, and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells as determined by teratoma formation assay.Conclusions/significanceThese findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells, and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells.
| S-EPMC2500174 | biostudies-literature
Project description:We earlier documented that lovastatin (LOV)-mediated inhibition of small Rho GTPases activity protects vulnerable oligodendrocytes (OLs) in mixed glial cell cultures stimulated with Th1 cytokines and in a murine model of multiple sclerosis (MS). However, the precise mechanism of OL protection remains unclear. We here employed genetic and biochemical approaches to elucidate the underlying mechanism that protects LOV treated OLs from Th1 (tumor necrosis factor-α) and Th17 (interleukin-17) cytokines toxicity in in vitro. Cytokines enhanced the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization with corresponding lowering of glutathione (reduced) level in OLs and that were reverted by LOV. In addition, the expression of ROS detoxifying enzymes (catalase and superoxide-dismutase 2) and the transactivation of peroxisome proliferators-activated receptor (PPAR)-α/-β/-γ including PPAR-γ coactivator-1α were enhanced by LOV in similarly treated OLs. Interestingly, LOV-mediated inhibition of small Rho GTPases, i.e., RhoA and cdc42, and Rho-associated kinase (ROCK) activity enhanced the levels of PPAR ligands in OLs via extracellular signal regulated kinase (1/2)/p38 mitogen-activated protein kinase/cytoplasmic phospholipase 2/cyclooxygenase-2 signaling cascade activation. Small hairpin RNA transfection-based studies established that LOV mainly enhances PPAR-α and less so of PPAR-β and PPAR-γ transactivation that enhances ROS detoxifying defense in OLs. In support of this, the observed LOV-mediated protection was lacking in PPAR-α-deficient OLs exposed to cytokines. Collectively, these data provide unprecedented evidence that LOV-mediated inhibition of the Rho-ROCK signaling pathway boosts ROS detoxifying defense in OLs via PPAR-α-dependent mechanism that has implication in neurodegenerative disorders including MS.
| S-EPMC3919553 | biostudies-literature
Project description:The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.
| S-EPMC3730436 | biostudies-literature
Project description:Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, DRF3, has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.
| S-EPMC3938721 | biostudies-literature
Project description:The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is known about the role of Cdc42 during normal mammary epithelial cell (MEC) morphogenesis. Here we aimed to define the precise role for Cdc42 during primary mammary acinus formation in vitro. For these studies, MECs were isolated from Cdc42fl/fl conditional knockout mice, transduced with Adeno-cre-GFP virus to delete Cdc42 or Adeno-GFP control virus, and effects on morphogenesis were investigated using a three-dimensional (3D) culture assay. Interestingly, markedly fewer mammary acini developed in Cdc42 deficient cultures, and the acini that formed were significantly smaller and disorganized. Cellular proliferation and survival were reduced in the Cdc42 deficient acini. However, control and knockout MECs cultured as monolayers displayed similar cell cycle profiles, suggesting that Cdc42 is important for MEC proliferation in the context of 3D polarity. Overexpression of cyclin D1, which promotes cell cycle progression downstream of Cdc42, failed to rescue the defect in acinus size. Furthermore, lumen formation and apical-basal polarity were disrupted, and mitotic spindle orientation and Cdc42/aPKC polarity complex defects likely contributed to these phenotypes. Studies using dominant negative Cdc42 and siRNa to knockdown Cdc42 in MDcK and Caco-2 cell lines undergoing cystogenesis in 3D cultures revealed critical roles for Cdc42 in spindle orientation, polarity and lumen formation. Our studies, using complete knockout in primary epithelial cells, demonstrate that Cdc42 is not only an important regulator of polarity and lumen formation; it is also essential for proliferation and survival, which are key cellular processes that drive MEC morphogenesis in vitro and in vivo.
| S-EPMC3265815 | biostudies-other
Project description:Fibroblast growth factor (FGF) signaling is essential for vertebrate organogenesis, including mammary gland development. The mechanism whereby FGF signaling is regulated in the mammary gland, however, has remained unknown. Using a combination of mouse genetics and 3D ex vivo models, we tested the hypothesis that Spry2 gene, which encodes an inhibitor of signaling via receptor tyrosine kinases (RTKs) in certain contexts, regulates FGF signaling during mammary branching. We found that Spry2 is expressed at various stages of the developing mammary gland. Targeted removal of Spry2 function from mammary epithelium leads to accelerated epithelial invasion. Spry2 is up-regulated by FGF signaling activities and its loss sensitizes mammary epithelium to FGF stimulation, as indicated by increased expression of FGF target genes and epithelia invasion. By contrast, Spry2 gain-of-function in the mammary epithelium results in reduced FGF signaling, epithelial invasion, and stunted branching. Furthermore, reduction of Spry2 expression is correlated with tumor progression in the MMTV-PyMT mouse model. Together, the data show that FGF signaling modulation by Spry2 is essential for epithelial morphogenesis in the mammary gland and it functions to protect the epithelium against tumorigenesis.
| S-EPMC3981693 | biostudies-literature
Project description:The genetic control of mammalian epithelial polarity and dynamics can be studied in vivo at cellular resolution during morphogenesis of the mouse neural tube. The mouse neural plate is a simple epithelium that is transformed into a columnar pseudostratified tube over the course of ∼ 24 h. Apical F-actin is known to be important for neural tube closure, but the precise roles of actin dynamics in the neural epithelium are not known. To determine how the organization of the neural epithelium and neural tube closure are affected when actin dynamics are blocked, we examined the cellular basis of the neural tube closure defect in mouse mutants that lack the actin-severing protein cofilin 1 (CFL1). Although apical localization of the adherens junctions, the Par complex, the Crumbs complex and SHROOM3 is normal in the mutants, CFL1 has at least two distinct functions in the apical and basal domains of the neural plate. Apically, in the absence of CFL1 myosin light chain does not become phosphorylated, indicating that CFL1 is required for the activation of apical actomyosin required for neural tube closure. On the basal side of the neural plate, loss of CFL1 has the opposite effect on myosin: excess F-actin and myosin accumulate and the ectopic myosin light chain is phosphorylated. The basal accumulation of F-actin is associated with the assembly of ectopic basal tight junctions and focal disruptions of the basement membrane, which eventually lead to a breakdown of epithelial organization.
| S-EPMC4378245 | biostudies-literature