Regulation Of Cytoskeleton Dynamics Research Articles

YAP\u2013TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP–TEAD respond to cell–cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell–cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP–TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP–TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP–TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

MTOR plays a pivotal role in multiple processes of enamel organ development principally through the mTORC1 pathway and in part via regulating cytoskeleton dynamics

We herein report that deletion of mTOR in dental epithelia caused defective development of multiple cell layers of the enamel organ, which culminated in tooth malformation and cystogenesis. Specifically, cells of the stellate reticulum and stratum intermedium were poorly formed, resulting in cystic changes. The pre-ameloblasts failed to elongate along the apical-basal axis and persisted vigorous expression of Sox2 and P63, which are normally downregulated during cytodifferentiation. Expression of amelogenic markers was also attenuated in mutants. Cell proliferation and cell sizes in mutants were significantly reduced over time. Importantly, we found reduced amounts and aberrant aggregations of cytoskeletal components in mutants, along with attenuated expression of cytoskeleton regulator Cdc42, whose epithelial deletion causes a similar phenotype. Moreover, disruption of actin assembly in an organ culture system affected cell proliferation and cytodifferentiation of tooth germs, supporting a causative role of mTOR-regulated cytoskeleton dynamics for the observed phenotype of mTOR mutant mice. In further support of this view, we showed that mTOR overactivation caused increased cytoskeletal component synthesis and assembly, along with accelerated cytodifferentiation in the enamel organ. Finally, we demonstrated that mTOR regulated enamel organ development principally through the mTORC1 pathway.

Abstract 4907: A novel functional analysis of MET exon 14 skipping mutation in regulation of tumor cell invasion and metastasis

Abstract MET exon 14 skipping mutations (METΔ14Ex) represent a new class of molecular alterations that occur in approximately 3%-5% of non-small cell lung cancer (NSCLC) cases, and recently emerged as an actionable oncogenic driver with significant responsiveness to MET tyrosine kinase inhibitors, including crizotinib and capmatinib. METΔ14Ex mutations are generally was found to be mutually exclusive with other recognized oncogenic drivers and higher frequency has been noted in a unique, highly aggressive subtype of lung cancer called pulmonary sarcomatoid carcinoma. In the current study, we sought to perform an array of experiments to determine the intrinsic tumorigenic role of METΔ14Ex in NSCLC. Using multiple isogenic METΔ14Ex cell models established with CRISPR, we first determined that METΔ14Ex increased HGF-dependent kinase activity and enhanced HGF/MET downstream signaling through impairing receptor endocytic degradation. In subsequent functional analyses, we identified that METΔ14Ex significantly increases HGF-dependent cell scatter, migration and invasion capacity in vitro as well as metastasis in vivo compared to wild-type MET (MET WT). To explore the intrinsic mechanism in regulation of aforementioned phenotypes, an RNA sequencing study was conducted to evaluate the global changes of dynamic gene expression profile and key functional pathways. Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) revealed a large cluster of cell movement-related signaling pathways that were remarkably activated in HGF-treated METΔ14Ex models (vs MET WT) in a time-dependent manner. In addition, a number of upregulated DEGs (Differently Expressed Genes) were enriched in functional categories of cell cytoskeleton reorganization and cell-matrix adhesion degradation in METΔ14Ex models (vs MET WT). Based on the information of GSEA, we further found that the activity of the small GTPase Rac1, a crucial regulator of cytoskeleton dynamics, was significantly increased by HGF treatment in METΔ14Ex cells, whereas inhibition of the PI3K/Akt pathway repressed HGF/METΔ14Ex-mediated Rac1 activity and cell movement. Moreover, a group of Matrix metallopeptidases (MMPs) were highly upregulated in our models. Notably, among these MMPs, HGF-dependent upregulation of MMP1 expression (>100 fold in both mRNA and protein level) was very prominent in METΔ14Ex compared to MET WT cells, and silencing MMP1 using siRNA dramatically repressed HGF-triggered METΔ14Ex cell invasion. We finally found that the MET inhibitors, MGCD516 and capmatinib remarkably inhibited HGF/METΔ14Ex signaling and repressed cell invasion in vitro and metastasis in vivo. In conclusion, these data demonstrate that METΔ14Ex plays a critical role in enhancing cell movement and metastasis, at least partially via upregulation of the PI3K/Akt-Rac1 pathway and expression of MMPs, and blockade of HGF/METΔ14Ex signaling using MET inhibitors might be potent therapeutic options for METΔ14Ex-mutated NSCLC patients and recognition of the unique biology of METΔ14Ex-mediated oncogenesis might yield additional synergistic treatment strategies. Citation Format: Feng Wang, Yang Liu, Elaine Shum, Mariam Alexander, Deyou Zheng, Roman Perez-Soler, Haiying Cheng, Balazs Halmos. A novel functional analysis of MET exon 14 skipping mutation in regulation of tumor cell invasion and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4907.

Research progress on intrinsic signaling pathways in axon regeneration

The intrinsic regrowth ability of injured neurons is essential for axon regeneration and functional recovery. Recently, numerous intrinsic pathways that regulate axon regeneration have been discovered, among which the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway are arguably the best characterized examples. MAPK signaling pathway is involved in multiple processes including sensing injury signals, initiating and promoting axonal regrowth through regulating cytoskeleton dynamics and protein synthesis. The PI3K/Akt signaling pathway regulates axon regeneration mainly through gene transcription and translation. Combinatory manipulation of multiple regeneration-promoting signals can further improve the extend of axonal regrowth. This paper summarizes current progresses on axon regeneration studies in various organisms and discuss their potentials in promoting functional recovery in vivo.

Vangl2 acts at the interface between actin and N-cadherin to modulate mammalian neuronal outgrowth.

Dynamic mechanical interactions between adhesion complexes and the cytoskeleton are essential for axon outgrowth and guidance. Whether planar cell polarity (PCP) proteins, which regulate cytoskeleton dynamics and appear necessary for some axon guidance, also mediate interactions with membrane adhesion is still unclear. Here we show that Vangl2 controls growth cone velocity by regulating the internal retrograde actin flow in an N-cadherin-dependent fashion. Single molecule tracking experiments show that the loss of Vangl2 decreased fast-diffusing N-cadherin membrane molecules and increased confined N-cadherin trajectories. Using optically manipulated N-cadherin-coated microspheres, we correlated this behavior to a stronger mechanical coupling of N-cadherin with the actin cytoskeleton. Lastly, we show that the spatial distribution of Vangl2 within the growth cone is selectively affected by an N-cadherin-coated substrate. Altogether, our data show that Vangl2 acts as a negative regulator of axonal outgrowth by regulating the strength of the molecular clutch between N-cadherin and the actin cytoskeleton.

LOXL2 promotes oncogenic progression in alveolar rhabdomyosarcoma independently of its catalytic activity

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. Patients with the most aggressive histological variant have an unfavorable prognosis due to a high metastasis incidence. Lysyl oxidase-like 2 (LOXL2) is a lysyl oxidase, member of a family of extracellular matrix (ECM) crosslinking enzymes that recently have emerged as important regulators of tumor progression and metastasis. We report that LOXL2 is overexpressed in RMS, suggesting a potential role for LOXL2 in RMS oncogenic progression. Consistently, transient and stable LOXL2 knockdown decreased cell migratory and invasive capabilities in two ARMS cell lines. Furthermore, introduction of LOXL2 in RMS non-expressing cells using wild type or mutated (catalytically inactive) constructs resulted in increased cell migration, cell invasion and number and incidence of spontaneous lung metastasis in vivo, independently of its catalytic activity. To further study the molecular mechanism associated with LOXL2 expression, a pull-down assay on LOXL2-transfected cells was performed and analyzed by mass spectrometry. The intermediated filament protein vimentin was validated as a LOXL2-interactor. Thus, our results suggest an oncogenic role of LOXL2 in RMS by regulating cytoskeleton dynamics and cell motility capabilities.

The Rho GTPase RND3 regulates adipocyte lipolysis

BackgroundAdipose tissue plays a crucial role in diet- and obesity-related insulin resistance, with implications for several metabolic diseases. Identification of novel target genes and mechanisms that regulate adipocyte function could lead to improved treatment strategies. RND3 (RhoE/Rho8), a Rho-related GTP-binding protein that inhibits Rho kinase (ROCK) signaling, has been linked to diverse diseases such as apoptotic cardiomyopathy, heart failure, cancer and type 2 diabetes, in part by regulating cytoskeleton dynamics and insulin-mediated glucose uptake. ResultsWe here investigated the expression of RND3 in adipose tissue in human obesity, and discovered a role for RND3 in regulating adipocyte metabolism. In cross-sectional and prospective studies, we observed 5-fold increased adipocyte levels of RND3 mRNA in obesity, reduced levels after surgery-induced weight loss, and positive correlations of RND3 mRNA with adipocyte size and surrogate measures of insulin resistance (HOMA2-IR and circulating triglyceride/high-density lipoprotein cholesterol (TAG/HDL-C) ratio). By screening for RND3-dependent gene expression following siRNA-mediated RND3 knockdown in differentiating human adipocytes, we found downregulation of inflammatory genes and upregulation of genes related to adipocyte ipolysis and insulin signaling. Treatment of adipocytes with tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), hypoxia or cAMP analogs increased RND3 mRNA levels 1.5–2-fold. Functional assays in primary human adipocytes confirmed that RND3 knockdown reduces cAMP- and isoproterenol-induced lipolysis, which were mimicked by treating cells with ROCK inhibitor. This effect could partly be explained by reduced protein expression of adipose triglyceride lipase (ATGL) and phosphorylated hormone-sensitive lipase (HSL). ConclusionWe here uncovered a novel differential expression of adipose RND3 in obesity and insulin resistance, which may at least partly depend on a causal effect of RND3 on adipocyte lipolysis.

RAB35 depletion affects spindle formation and actin-based spindle migration in mouse oocyte meiosis.

Mammalian oocyte maturation involves a unique asymmetric cell division, in which meiotic spindle formation and actin filament-mediated spindle migration to the oocyte cortex are key processes. Here, we report that the vesicle trafficking regulator, RAB35 GTPase, is involved in regulating cytoskeleton dynamics in mouse oocytes. RAB35 GTPase mainly accumulated at the meiotic spindle periphery and cortex during oocyte meiosis. Depletion of RAB35 by morpholino microinjection led to aberrant polar body extrusion and asymmetric division defects in almost half the treated oocytes. We also found that RAB35 affected SIRT2 and αTAT for tubulin acetylation, which further modulated microtubule stability and meiotic spindle formation. Additionally, we found that RAB35 associated with RHOA in oocytes and modulated the ROCK-cofilin pathway for actin assembly, which further facilitated spindle migration for oocyte asymmetric division. Importantly, microinjection of Myc-Rab35 cRNA into RAB35-depleted oocytes could significantly rescue these defects. In summary, our results suggest that RAB35 GTPase has multiple roles in spindle stability and actin-mediated spindle migration in mouse oocyte meiosis.

PS1002 RHOE, CXCL12 AND CXCR3 MAY IDENTIFY COMPLETE RESPONSES IN ACUTE MYELOID LEUKEMIA PATIENTS TREATED WITH TIPIFARNIB

Background:RHOE is a uniquely farnesylated antagonist of RHOA that regulates cytoskeleton dynamics, cell mobilization and metalloproteinase (MMP)‐mediated tissue invasion. Recent data suggest that CXCL12 can be cleaved by MMPs generating a toxic fragment CXCL12(5–67) that no longer binds to its receptor CXCR4 but that can interact with CXCR3‐B and induce cell death.Aims:To characterize the mechanism of action of tipifarnib in AML, herein, we describe the identification of RHOE as a potential target of tipifarnib that could mediate CXCR3 activation by CXCL12 and response to farnesyl transferase inhibition in AML patients.Methods:CTEP20 was a phase 2, multicenter, open‐label study that investigated the efficacy and safety of the farnesyl transferase inhibitor tipifarnib in 158 older adults with previously untreated, poor‐risk AML. INT11 was a multicenter phase 2 study that evaluated tipifarnib in 252 pts with relapsed/refractory AML. Global gene expression data was generated using the Affymetrix U133A gene chip from 92 pretreatment BM samples from CTEP20 and INT11 pts (GSE8970, GSE5122) and analyzed retrospectively with respect to study outcomes. The Kaplan‐Meier method was employed to estimate survival and progression free survival (PFS) and the analysis of prognostic variables. Clinical trial information: NCT00027872, NCT00101296.Results:We have previously shown that CXCL12 expression determines bone marrow homing and sensitivity to tipifarnib in AML (Gualberto, ASH 2017). We recently identified RHOE in a screening of AML databases for genes of uniquely farnesylated proteins that are co‐expressed with CXCL12 (TCGA AML, provisional N = 163, r = 0.723, p < 0.0001). RHOE and CXCL12 gene expression were also strongly correlated with VCAM1 expression (p < 0.0001), suggesting stromal origin. We then investigated the activity of tipifarnib in AML patients with bone marrow expression of RHOE, CXCL12 and CXCR3. Analysis of bone marrow samples from AML patients enrolled in CTEP20 showed that pre‐treatment expression of CXCR3, CXCL12, and RHOE were highly significantly (p < 0.0001) predictive of complete response (CR) on tipifarnib treatment with ROC AUC values of 0.862, 0.782, and 0.736, respectively. The most discriminating index was the product of the expression of CXCL12 (ligand) and CXCR3 (receptor). The 80th percentile of the product of the expression of CXCL12 and CXCR3 had a 100% PPV and 94% NPV to identify a CR outcome, assuming a true 10% CR rate in the unselected population. PFS in the high CXCL12 x CXCR3 subset was 433 days versus 64 days in the low expressing subset (HR = 0.24, p = 0.005, N = 34). CXCR3 expression also predicted long term survival in study INT11. Median survival times in relapsed/refractory AML patients with CXCR3 expression tertiales 1st‐3rd were respectively 54, 75 and 182 days (p = 0.009, N = 58). Ex vivo treatment of bone marrow stromal cell cultures with tipifarnib showed loss of CXCL12 immunoreactivity and current work investigates its potential tipifarnib‐induced metabolism.Summary/Conclusion:These findings provide a potential RHOE‐mediated mechanism of action of tipifarnib in AML patients with bone marrow CXCL12 and CXCR3 expression.

Doublecortin X (DCX) serine 28 phosphorylation is a regulatory switch, modulating association of DCX with microtubules and actin filaments

Doublecortin X (DCX) plays essential roles in neuronal development via its regulation of cytoskeleton dynamics. This is mediated through direct interactions between its doublecortin (DC) domains (DC1 and DC2) with microtubules (MTs) and indirect association with actin filaments (F-ACT). While the regulatory role of the DCX C-terminus following DC2 (i.e. DCX residues 275–366) has been established, less is known of the possible contributions made by the DCX N-terminus preceding DC1 (i.e. DCX residues 1–44). Here, we assessed the influence of DCX Ser28 within the DCX N-terminus, on the association of DCX with MTs and F-ACT. We compared the cytoskeletal interactions of the DCX S28E phosphomimetic and DCX S28A phospho-resistant mutants and wild-type DCX. Immunoprecipitation and colocalisation analyses indicated increased association of DCX S28E with F-ACT but decreased interaction with MTs, and conversely enhanced DCX S28A association with MTs but decreased association with F-ACT. To evaluate the impact of DCX mutants on cytoskeletal filaments we performed fluorescence recovery after photobleaching (FRAP) studies on SiR-tubulin and β-actin-mCherry and observed comparable tubulin and actin exchange rates in the presence of DCX WT and DCX S28A. However, we observed faster tubulin exchange rates but slower actin exchange rates in the presence of DCX S28E. Moreover, DCX S28E enhanced the association with the actin-binding protein spinophilin (Spn) suggesting the shift to favour association with both F-ACT and Spn in the presence of DCX S28E. Taken together, our results highlight a new role for DCX S28 as a regulatory switch for cytoskeletal organisation.

Brain proteome changes in female Brd1+/− mice unmask dendritic spine pathology and show enrichment for schizophrenia risk

Genetic and molecular studies have implicated the Bromodomain containing 1 (BRD1) gene in the pathogenesis of schizophrenia and bipolar disorder. Accordingly, mice heterozygous for a targeted deletion of Brd1 (Brd1+/− mice) show behavioral phenotypes with broad translational relevance to psychiatric disorders. BRD1 encodes a scaffold protein that affects the expression of many genes through modulation of histone acetylation. BRD1 target genes have been identified in cell lines; however the impact of reduced Brd1 levels on the brain proteome is largely unknown. In this study, we applied label-based quantitative mass spectrometry to profile the frontal cortex, hippocampus and striatum proteome and synaptosomal proteome of female Brd1+/− mice. We successfully quantified between 1537 and 2196 proteins and show widespread changes in protein abundancies and compartmentalization. By integrative analysis of human genetic data, we find that the differentially abundant proteins in frontal cortex and hippocampus are enriched for schizophrenia risk further linking the actions of BRD1 to psychiatric disorders. Affected proteins were further enriched for proteins involved in processes known to influence neuronal and dendritic spine morphology e.g. regulation of cytoskeleton dynamics and mitochondrial function. Directly prompted in these findings, we investigated dendritic spine morphology of pyramidal neurons in anterior cingulate cortex and found them significantly altered, including reduced size of small dendritic spines and decreased number of the mature mushroom type. Collectively, our study describes known as well as new mechanisms related to BRD1 dysfunction and its role in psychiatric disorders, and provides evidence for the molecular and cellular dysfunctions underlying altered neurosignalling and cognition in Brd1+/− mice.

PAK2 Haploinsufficiency Results in Synaptic Cytoskeleton Impairment and Autism-Related Behavior

Synaptic cytoskeleton dysfunction represents a common pathogenesis in neurodevelopmental disorders, such as autism spectrum disorder (ASD). The serine/threonine kinase PAK2 is a critical regulator of cytoskeleton dynamics. However, its function within the central nervous system and its role in ASD pathogenesis remain undefined. Here, we found thatPak2 haploinsufficiency resulted in markedly decreased synapse densities, defective long-term potentiation, and autism-related behaviors in mice. Phosphorylation levels of key actin regulators LIMK1 and cofilin, together with their mediated actin polymerization, were reduced in Pak2+/-mice. We identified one de novo PAK2 nonsense mutation that impaired PAK2 function invitro and invivo and four de novo copy-number deletions containing PAK2 in large cohorts of patients with ASD. PAK2 deficiency extensively perturbed functional networks associated with ASD by regulating actin cytoskeleton dynamics. Our genetic and functional results demonstrate a critical role of PAK2 in brain development and autism pathogenesis.

Crawling migration under chemical signalling: A stochastic model

Cell migration is an essential and complex mechanism involved in many physiological processes such as tissue formation or tumor invasion. External and internal cues are known to guide cell motion by regulating cytoskeleton dynamics. Here, we approach cell‐crawling migration under a large‐scale assumption so that it reduces to a particle in motion. However, we describe the cell displacement as the result of its internal activity: the cell velocity is a function of the (discrete) protrusive forces exerted by filopodia on the substrate. Cell polarization ability is modeled in the feedback that the cell motion exerts on the protrusion rates: faster cells form preferentially protrusions in the direction of motion. In addition, we incorporate a gradient in attractive chemical signal, which may vary in time. We perform numerical simulations on this model, showing that the balance between cells self‐polarized internal machinery and signal sensing leads to nontrivial behaviors. Finally, by using the mathematical framework of structured population processes previously developed to study population dynamics, we study rigorously the mathematical model, and we derive some of its fundamental properties.

IRE1α governs cytoskeleton remodelling and cell migration through a direct interaction with filamin A.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a signalling network known as the unfolded protein response (UPR). Here, we identified filamin A as a major binding partner of the ER stress transducer IRE1α. Filamin A is an actin crosslinking factor involved in cytoskeleton remodelling. We show that IRE1α controls actin cytoskeleton dynamics and affects cell migration upstream of filamin A. The regulation of cytoskeleton dynamics by IRE1α is independent of its canonical role as a UPR mediator, serving instead as a scaffold that recruits and regulates filamin A. Targeting IRE1α expression in mice affected normal brain development, generating a phenotype resembling periventricular heterotopia, a disease linked to the loss of function of filamin A. IRE1α also modulated cell movement and cytoskeleton dynamics in fly and zebrafish models. This study unveils an unanticipated biological function of IRE1α in cell migration, whereby filamin A operates as an interphase between the UPR and the actin cytoskeleton.

The dosage-dependent effect exerted by the NM23-H1/H2 homolog NDK-1 on distal tip cell migration in C. elegans

Abnormal regulation of cell migration and altered rearrangement of the cytoskeleton are fundamental properties of metastatic cells. The first identified metastasis suppressor NM23-H1, which displays nucleoside-diphosphate kinase (NDPK) activity is involved in these processes. NM23-H1 inhibits the migratory and invasive potential of some cancer cells. Correspondingly, numerous invasive cancer cell lines (eg, breast, colon, oral, hepatocellular carcinoma, and melanoma) display low endogenous NM23 levels. In this review, we summarize mechanisms, which are linked to the anti-metastatic activity of NM23. In human cancer cell lines NM23-H1 was shown to regulate cytoskeleton dynamics through inactivation of Rho/Rac-type GTPases. The Drosophila melanogaster NM23 homolog abnormal wing disc (AWD) controls tracheal and border cell migration. The molecular function of AWD is well characterized in both processes as a GTP supplier of Shi/Dynamin whereby AWD regulates the level of chemotactic receptors on the surface of migrating cells through receptor internalization, by its endocytic function. Our group studied the role of the sole group I NDPK, NDK-1 in distal tip cell (DTC) migration in Caenorhabditis elegans. In the absence of NDK-1 the migration of DTCs is incomplete. A half dosage of NDPK as present in ndk-1 (+/−) heterozygotes results in extra turns and overshoots of migrating gonad arms. Conversely, an elevated NDPK level also leads to incomplete gonadal migration owing to a premature stop of DTCs in the third phase of migration, where NDK-1 acts. We propose that NDK-1 exerts a dosage-dependent effect on the migration of DTCs. Our data derived from DTC migration in C. elegans is consistent with data on AWD's function in Drosophila. The combined data suggest that NDPK enzymes control the availability of surface receptors to regulate cell-sensing cues during cell migration. The dosage of NDPKs may be a coupling factor in cell migration by modulating the efficiency of receptor recycling.

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling.

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions.

TCR signalling network organization at the immunological synapses of murine regulatory T cells.

Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of Tcell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.

Sirtuin 2 Deficiency Increases Bacterial Phagocytosis by Macrophages and Protects from Chronic Staphylococcal Infection.

Sirtuin 2 (SIRT2) is one of the seven members of the family of NAD+-dependent histone deacetylases. Sirtuins target histones and non-histone proteins according to their subcellular localization, influencing various biological processes. SIRT2 resides mainly in the cytoplasm and regulates cytoskeleton dynamics, cell cycle, and metabolic pathways. As such, SIRT2 has been implicated in the pathogenesis of neurodegenerative, metabolic, oncologic, and chronic inflammatory disorders. This motivated the development of SIRT2-directed therapies for clinical purposes. However, the impact of SIRT2 on antimicrobial host defense is largely unknown. Here, we address this question using SIRT2 knockout mice. We show that SIRT2 is the most highly expressed sirtuin in myeloid cells, especially macrophages. SIRT2 deficiency does not affect immune cell development and marginally impacts on intracellular signaling and cytokine production by splenocytes and macrophages. However, SIRT2 deficiency enhances bacterial phagocytosis by macrophages. In line with these observations, in preclinical models, SIRT2 deficiency increases survival of mice with chronic staphylococcal infection, while having no effect on the course of toxic shock syndrome toxin-1, LPS or TNF-induced shock, fulminant Escherichia coli peritonitis, sub-lethal Klebsiella pneumoniae pneumonia, and chronic candidiasis. Altogether, these data support the safety profile of SIRT2 inhibitors under clinical development in terms of susceptibility to infections.

AMP-activated protein kinase/myocardin-related transcription factor-A signaling regulates fibroblast activation and renal fibrosis

Chronic kidney disease is a major cause of death, and renal fibrosis is a common pathway leading to the progression of this disease. Although activated fibroblasts are responsible for the production of the extracellular matrix and the development of renal fibrosis, the molecular mechanisms underlying fibroblast activation are not fully defined. Here we examined the functional role of AMP-activated protein kinase (AMPK) in the activation of fibroblasts and the development of renal fibrosis. AMPKα1 was induced in the kidney during the development of renal fibrosis. Mice with global or fibroblast-specific knockout of AMPKα1 exhibited fewer myofibroblasts, developed less fibrosis, and produced less extracellular matrix protein in the kidneys following unilateral ureteral obstruction or ischemia-reperfusion injury. Mechanistically, AMPKα1 directly phosphorylated cofilin leading to cytoskeleton remodeling and myocardin-related transcription factor-A nuclear translocation resulting in fibroblast activation and extracellular matrix protein production. Thus, AMPK may be a critical regulator of fibroblast activation through regulation of cytoskeleton dynamics and myocardin-related transcription factor-A nuclear translocation. Hence, AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.

HGF/Met Signaling in Cancer Invasion: The Impact on Cytoskeleton Remodeling.

The invasion of cancer cells into surrounding tissue and the vasculature is essential for tumor metastasis. Increasing evidence indicates that hepatocyte growth factor (HGF) induces cancer cell migration and invasion. A broad spectrum of mechanisms underlies cancer cell migration and invasion. Cytoskeletal reorganization is of central importance in the development of the phenotype of cancer cells with invasive behavior. Through their roles in cell mechanics, intracellular trafficking, and signaling, cytoskeleton proteins participate in all essential events leading to cell migration. HGF has been involved in cytoskeleton assembly and reorganization, and its role in regulating cytoskeleton dynamics is still expanding. This review summarizes our current understanding of the role of HGF in regulating cytoskeleton remodeling, distribution, and interactions.