Cytoskeleton Remodeling Research Articles

Combined dynamic transcriptomics and metabolomics analyses revealed the effects of trans-vp28 gene Synechocystis sp. PCC6803 on the hepatopancreas of Litopenaeus vannamei

Litopenaeus vannamei is the most important shrimp species throughout the world. However, diseases are increasing with the development of the industry, so enhancing the immunity of shrimp is of great significance. In this study, 1800 shrimp were divided into two groups randomly: the control group (N, feed with brine shrimp flake) and the experimental group (M, feed with mutant of Synechocystis sp. cells) (300 shrimp/group/replication) and each trial was conducted in triplicates. After immunization, sixty shrimp (with three replicates of twenty) were collected at 0 h in group N and 24, 72, and 144 h in group M, respectively, and the hepatopancreas were isolated for transcriptomic and metabolomic analysis. Transcriptome data revealed that compared with group N, genes related to antimicrobial peptides, cytoskeleton remodeling, detoxification, apoptosis, blood coagulation, immune defense, and antioxidant systems were differentially expressed in group M. In addition, combined transcriptomic and metabolomic analysis revealed that some immune-related differential genes or differential metabolites were consistently expressed in both omics. All the above results indicated that trans-vp28 gene Synechocystis sp. PCC6803 could improve the immunity of L. vannamei.This is the first report of the integration of dynamic transcriptomics combined with metabolomics to study the effect of trans-vp28 gene Synechocystis sp. PCC6803 in the hepatopancreas of L. vannamei and provided important information about the defense and immune mechanisms used by invertebrates against pathogens.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

High Concentrations of Genistein Decrease Cell Viability Depending on Oxidative Stress and Inflammation in Colon Cancer Cell Lines.

Genistein could play a crucial role in modulating three closely linked physiological processes altered during cancer: oxidative stress, mitochondrial biogenesis, and inflammation. However, genistein’s role in colorectal cancer remains unclear. We aimed to determine genistein’s effects in two colon cancer cells: HT29 and SW620, primary and metastatic cancer cells, respectively. After genistein treatment for 48 h, cell viability and hydrogen peroxide (H2O2) production were studied. The cell cycle was studied by flow cytometry, mRNA and protein levels were analyzed by RT-qPCR and Western blot, respectively, and finally, cytoskeleton remodeling and NF-κB translocation were determined by confocal microscopy. Genistein 100 µM decreased cell viability and produced G2/M arrest, increased H2O2, and produced filopodia in SW620 cells. In HT29 cells, genistein produced an increase of cell death, H2O2 production, and in the number of stress fibers. In HT29 cells, mitochondrial biogenesis was increased, however, in SW620 cells, it was decreased. Finally, the expression of inflammation-related genes increased in both cell lines, being greater in SW620 cells, where NF-κB translocation to the nucleus was higher. These results indicate that high concentrations of genistein could increase oxidative stress and inflammation in colon cancer cells and, ultimately, decrease cell viability.

真核细胞运动接触抑制行为的力学-化学耦合模拟

Contact inhibition of locomotion (CIL) drives various biological phenomena, including cell dispersion, collective cell migration, and cancer invasion. Based on our previously proposed cell migration model with multilayered signaling cascades, a mechanical-chemical coupling model for CIL was developed by further incorporating cell-cell contact-dependent signaling pathways to guide the cytoskeleton remodeling. The cell structure model incorporates a discrete actin filament network within a cycle. The entire mathematical model is composed of nonlinear diffusion-reaction equations coupled with force-balance equations and solved numerically by the in-house built LBP-D1Q3 method. Numerical simulations indicated that the manifestation of distinct CIL behavior depends on the cooperation between FilGAP-FLNa and N-cadherin signaling pathways in terms of Rho GTPase signaling. After achieving a suitable inhibitory effect of FilGAP, the cells retain moderate polarity, which can be reversed in response to N-cadherin signaling, leading to normal CIL behavior. The lack of FilGAP transforms the cells to a fully polarized state, which is difficult to reverse, thus leading to the “bypass” behavior. The lack of N-cadherin signaling prevents cells from repolarizing in response to cell-cell contact, leading to static cell-cell adhesion. These simulations provide a theoretical basis for understanding the effect of contact inhibition signaling on cell migration.

From Transcriptomics, Metabolomics to Functional Studies: Extracellular ATP Induces TGF-β-Like Epithelial Mesenchymal Transition in Lung Cancer Cells.

We and others previously showed that extracellular ATP (eATP) is implicated in epithelial mesenchymal transition (EMT). However, the mechanisms by which eATP induces EMT and ATP’s relationship to TGF-β, a well-known EMT inducer, are largely unclear. Also, eATP-induced EMT has never been studied at transcriptomic and metabolomics levels. Based on our previous studies, we hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels in cancer cells. RNAseq and metabolomics analyses were performed on human non-small cell lung cancer (NSCLC) A549 cells treated with either eATP or TGF-β. Bio-functional assays, such as invasion, intracellular ATP, cell proliferation, cytoskeleton remodeling, and others were conducted in NSCLC A549 and H1299 cells to validate changes observed from RNAseq and metabolomics studies. In the RNAseq study, eATP significantly enriched expressions of genes involved in EMT similarly to TGF-β after 2 and 6 hours of treatment. Samples treated with eATP for 2 hours share 131 upregulated EMT genes with those of TGF-β treated samples, and 42 genes at 6 hours treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. BLOC1S6, one of the 11 genes, was selected for further study. eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar to those induced by TGF-β. Functional bioassays verified the findings from RNAseq and metabolomics that eATP EMT-like changes in A549 and H1299 cells similarly to TGF-β. BLOC1S6 was found to be implicated in EMT. In these studies, eATP-induced EMT, at all levels examined, is similar but non-identical to that induced by TGF-β, and functions in such a way that exogenous addition of TGF-β is unnecessary for the induction. The study of BLOC1S6 further verified its potential roles in EMT and the RNAseq analysis results. All these strongly indicate that eATP is a multi-functional and multi-locational inducer and regulator of EMT, changing our thinking on how EMT is induced and regulated and pointing to new directions for inhibiting EMT in cancer.

Obstacles to Glioblastoma Treatment Two Decades after Temozolomide.

Simple SummaryGlioblastomas are the most common and aggressive brain tumors in adults, with a median survival of 15 months. Treatment is surgical removal, followed by chemotherapy and/or radiotherapy. Current chemotherapeutics do not kill all the tumor cells and some cells survive, leading to the appearance of a new tumor resistant to the treatment. These treatment-resistant cells are called tumor stem cells. In addition, glioblastoma cells have a high capacity for migration, forming new tumors in areas distant from the original tumor. Studies are now focused on understanding the molecular mechanisms of chemoresistance and controlling drug entry into the brain to improve drug performance. Another promising therapeutic approach is the use of viruses that specifically destroy glioblastoma cells, preserving the neural tissue around the tumor. In this review, we summarize the main biological features of glioblastoma and the therapeutic targets that are currently under study for new clinical trials.Glioblastomas are considered the most common and aggressive primary brain tumor in adults, with an average of 15 months’ survival rate. The treatment is surgery resection, followed by chemotherapy with temozolomide, and/or radiotherapy. Glioblastoma must have wild-type IDH gene and some characteristics, such as TERT promoter mutation, EGFR gene amplification, microvascular proliferation, among others. Glioblastomas have great heterogeneity at cellular and molecular levels, presenting distinct phenotypes and diversified molecular signatures in each tumor mass, making it difficult to define a specific therapeutic target. It is believed that the main responsibility for the emerge of these distinct patterns lies in subcellular populations of tumor stem cells, capable of tumor initiation and asymmetric division. Studies are now focused on understanding molecular mechanisms of chemoresistance, the tumor microenvironment, due to hypoxic and necrotic areas, cytoskeleton and extracellular matrix remodeling, and in controlling blood brain barrier permeabilization to improve drug delivery. Another promising therapeutic approach is the use of oncolytic viruses that are able to destroy specifically glioblastoma cells, preserving the neural tissue around the tumor. In this review, we summarize the main biological characteristics of glioblastoma and the cutting-edge therapeutic targets that are currently under study for promising new clinical trials.

Protein kinase C-β distinctly regulates blood-brain barrier-forming capacity of Brain Microvascular endothelial cells and outgrowth endothelial cells

Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-β (PKC-β) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-β inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-β protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-β signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.

Actin remodelling controls proteasome homeostasis upon stress

When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins are simultaneously upregulated. To promote proteasome assembly and activity and maintain cell viability upon TORC1 inhibition, 19S regulatory-particle assembly chaperones (RPACs) are selectively translated. However, the molecular mechanism for such selective translational upregulation is unclear. Here, using yeast, we discover that remodelling of the actin cytoskeleton is important for RPAC translation following TORC1 inhibition. mRNA of the RPAC ADC17 is associated with actin cables and is enriched at cortical actin patches under stress, dependent upon the early endocytic protein Ede1. ede1∆ cells failed to induce RPACs and proteasome assembly upon TORC1 inhibition. Conversely, artificially tethering ADC17 mRNA to cortical actin patches enhanced its translation upon stress. These findings suggest that actin-dense structures such as cortical actin patches may serve as a translation platform for a subset of stress-induced mRNAs including regulators of proteasome homeostasis.

Long non-coding RNA HOTAIR regulates cytoskeleton remodeling and lipid storage capacity during adipogenesis

The long non-coding RNA HOTAIR is the most differentially expressed gene between upper- and lower-body adipose tissue, yet its functional significance in adipogenesis is unclear. We report that HOTAIR expression is transiently induced during early adipogenic differentiation of gluteofemoral adipose progenitors and repressed in mature adipocytes. Upon adipogenic commitment, HOTAIR regulates protein synthesis pathways and cytoskeleton remodeling with a later impact on mature adipocyte lipid storage capacity. Our results support novel and important functions of HOTAIR in the physiological context of adipogenesis.

Impact of Surface Chemistry of Ultrasmall Superparamagnetic Iron Oxide Nanoparticles on Protein Corona Formation and Endothelial Cell Uptake, Toxicity, and Barrier Function.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) have been investigated for biomedical applications, including novel contrast agents, magnetic tracers for tumor imaging, targeted drug delivery vehicles, and magneto-mechanical actuators for hyperthermia and thrombolysis. Despite significant progress, recent clinical reports have raised concerns regarding USPION safety related to endothelial cell dysfunction; however, there is limited information on factors contributing to these clinical responses. The influence of USPION surface chemistry on nanoparticle interactions with proteins may impact endothelial cell function leading to adverse responses. Therefore, the goal of this study was to assess the effects of carboxyl-functionalized USPION (CU) or amine-functionalized USPION (AU) (approximately 30 nm diameter) on biological responses in human coronary artery endothelial cells. Increased protein adsorption was observed for AU compared with CU after exposure to serum proteins. Exposure to CU, but not AU, resulted in a concentration-dependent decrease in cell viability and perinuclear accumulation inside cytoplasmic vesicles. Internalization of CU was correlated with endothelial cell functional changes under non-cytotoxic conditions, as evidenced by a marked decreased expression of endothelial-specific adhesion proteins (eg, vascular endothelial-cadherin and platelet endothelial cell adhesion molecule-1) and increased endothelial permeability. Evaluation of downstream signaling indicated endothelial permeability is associated with actin cytoskeleton remodeling, possibly elicited by intracellular events involving reactive oxygen species, calcium ions, and the nanoparticle cellular uptake pathway. This study demonstrated that USPION surface chemistry significantly impacts protein adsorption and endothelial cell uptake, viability, and barrier function. This information will advance the current toxicological profile of USPION and improve development, safety assessment, and clinical outcomes of USPION-enabled medical products.

Abstract 5498: Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival

Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy. Aggressive, diffuse invasion is regulated by a dynamic cytoskeleton and significantly contributes to poor survival. Intraparenchymal tumor cell dissemination prevents complete surgical resection and compromises essential brain functions. Invasive cells are inherently less sensitive to traditional cytotoxic therapies. Recent investigation into targeted therapies has failed to address invasion/cytoskeleton based GBM therapy resistance. Furthermore, established GBM cell line modeling in 2 or 3 dimensions (3D) does not capture the genetic diversity or clinical relevance of patient derived primary (1') GBM cell lines, which may uncover/predict differential responses in tumors to novel therapies. Cytoskeleton remodeling and cell invasion in patient-derived primary GBM cells generates unique structures called tumor microtubes (TMs) not seen in established, cultured GBM cell lines. TMs are ultralong, pro-invasive, tubulin-enriched protrusions that promote chemo- and radioresistance in GBM patient derived primary cells. Using our extensive GBM cell line biobank derived from 1' and/or therapy resistant recurrent (2') tumors, we investigated the effects of a novel drug targeting the microtubule cytoskeleton. Plinabulin is a tubulin targeting agent that crosses the blood brain barrier and demonstrated anticancer efficacy in Phase III trial patients with non-small cell lung carcinoma. Plinabulin also has antiproliferative properties in 2D GBM cells. Here, we evaluate the efficacy of plinabulin treatment upon 3D neurosphere viability and invasion in 5 GBM 1' and 2' recurrent patient derived cell lines. Within 24h of matrigel embedding, 3D GBM patient neurospheres generate robust pro-invasive TMs. Plinabulin treatment (10-30 nM) at embedding followed by drug washout after 24h eliminated existing TMs and inhibited single cell invasion (>80% relative to controls) in both 1' and 2' GBM patient neurospheres. Cell survival was significantly reduced in plinabulin treated neurospheres (>30 nM). Plinabulin efficacy was evaluated in a clinically relevant therapeutic ITR model: GBM patient neurospheres invaded (I) for 48h to establish TM networks; were treated (T) with 1-50 nM plinabulin for 24h prior to drug washout; and recovered (R) for 96h. In 1', 2', or matched 1' and 2' cell line pairs from the same patient, 10-40 nM plinabulin sustained invasion inhibition (>90%) through 96h post washout and completely blocked TM extension relative to controls, consistent with established clinical concentrations of plinabulin. This study reveals plinabulin as a potent GBM therapeutic targeting pro-invasion TM networks and survival in 1' and therapy resistant 2' GBM patient cell lines. Citation Format: Kathryn M. Eisenmann, Kenneth Lloyd, Krista M. Pettee, Kathryn N. Becker, Jason Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlal, Lan Huang. Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5498.

Abstract 1365: BHLHE40 orchestrates remodeling of the intratumoral immune cell populations in response to immune checkpoint therapy

Abstract The success of immune checkpoint therapy (ICT) in generating durable clinical responses is remarkable but not all cancer patients respond for reasons that are incompletely understood. In our previous studies of immune responses to ICT in the mouse T3 sarcoma model, we found strong upregulation of the BHLHE40 transcription factor in a number of immune cell populations. It was particularly prominent in the tumor antigen-specific CD8+ and CD4+ T cells, which are crucial for ICT-induced tumor rejection and elimination. We used global and cell-type specific Bhlhe40 knockout (KO) mice to investigate its role in tumor rejection. Both global Bhlhe40-/- and CD4-Cre+ Bhlhe40f/f (CD4+ and CD8+ T cell-specific Bhlhe40 deletion) mice bearing the 1956 methylcholanthrene-induced sarcoma line failed to respond to either anti-PD-1 or anti-CTLA-4 ICT, in stark contrast to wild type (WT) mice. At the same time, Bhlhe40 deletion in macrophages and granulocytes (LysM-Cre+ Bhlhe40f/f mice) did not have an apparent effect on ICT-mediated tumor rejection. To gain insight into the functional changes orchestrated by BHLHE40 in response to ICT, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptional profiles of the CD45+ tumor-infiltrating immune cells from 1956 tumor-bearing Bhlhe40+/+ and Bhlhe40-/- mice treated with control, anti-PD-1 or anti-CTLA-4 mAb. While no major changes in the relative fractions of effector T cell clusters were observed, ICT induced BHLHE40-dependent remodeling within individual CD4+ and CD8+ clusters. Most notably, Bhlhe40-/- mice were strongly deficient in ICT-induced Ifng expression by CD8+ T cells, with intracellular cytokine staining revealing low IFNγ production by both CD4+ and CD8+ T cells. GSEA and Ingenuity Pathway analysis indicated changes in energy metabolism, hypoxia adaptation, NF-κB signaling, IFNγ response, and actin cytoskeleton remodeling in a number of Bhlhe40-/- T cell clusters. This was accompanied by upregulation of the inhibitory receptor Tigit mRNA, altered expression of chemokine/chemokine receptor genes, as well as granzyme gene family members. In addition to changes in the transcriptional profile shared between anti-PD-1 and anti-CTLA-4 treatments, each checkpoint inhibitor induced its own specific alterations both in WT and KO mice. Analysis of macrophage populations also revealed a notable difference between Bhlhe40+/+ and Bhlhe40-/- mice. The shift of macrophages from the CX3CR1+ CD206+ to the iNOS+ phenotype, typically observed during effective ICT, was dramatically reduced in the Bhlhe40-/- mice, which was confirmed by flow cytometry. Our results provide the basis for understanding the role of BHLHE40 in the ICT-induced tumor rejection, the pathways and effector mechanisms under its control, and the potential ways for therapeutic targeting of this regulatory network to improve the efficacy of cancer immunotherapy. Citation Format: Alexander S. Shavkunov, Avery J. Salmon, Qi Miao, Sunita Keshari, Charmelle D. Williams, Josué E. Pineda, Ken Chen, Brian T. Edelson, Matthew M. Gubin. BHLHE40 orchestrates remodeling of the intratumoral immune cell populations in response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1365.

Abstract 1578: Interaction of E-cadherin and EGFR in the collective cell migration of colon cancer cells

Abstract Collective cell migration is one of the most important cellular events where a cluster of cells moves together in a coordinated way. Many studies have found this phenomenon as the hallmark of cancer progression and metastasis. In epithelial single cell migration, downregulation of adherens junction (AJ) molecule E-cadherin occurs at the initial step known as the Epithelial to Mesenchymal Transition (EMT). However, in collective cell migration, E-cadherin junction is maintained to provide adhesion between cells. Moreover, it also mediates the cell-cell intercommunication that is critical, for instance, to alter the group polarization through actin cytoskeleton remodeling. Interestingly, Epidermal Growth Factor Receptor (EGFR) was found to colocalize with E-cadherin in the cell-cell contact region, implicating that E-cadherin and EGFR might interact in the cell junction of collective cell migration. To address this, a human colorectal cancer cell line exhibiting epithelial phenotype HT-29 was used. The cell density and calcium presence were modified to infer the effect of differential expression and stability of E-cadherin on the cellular signaling of proliferation and migration. The results showed that stable and enhanced expression of E-cadherin downregulates EGFR expression and reduces its activity. Moreover, the Src phosphorylation increased when the cell-cell contact was strengthened via calcium addition and decreased when the cell-cell contact was diminished via calcium chelation. Overall, this indicates that E-cadherin expression at the cell-cell junction and contact stability determine the EGFR expression, activity, and Src phosphorylation. With the goal of deciphering the relationship of E-cadherin and EGFR in cell contact, proliferation, and migration, we intend to finally implement the knowledge as a strategy or approach in promoting alternative methods to inhibit metastasis. Citation Format: Viriya Adhiguna Winarso, Yi-Wen Lu, Yu-Cheng Weng, Xiang-Ling Hou, Hui Min Koo, Yu-n Li, Wei-Ting Chao. Interaction of E-cadherin and EGFR in the collective cell migration of colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1578.

The focal adhesion protein β-parvin controls cardiomyocyte shape and sarcomere assembly in response to mechanical load

Physiological and pathological cardiac stress induced by exercise and hypertension, respectively, increase the hemodynamic load for the heart and trigger specific hypertrophic signals in cardiomyocytes leading to adaptive or maladaptive cardiac hypertrophy responses involving a mechanosensitive remodeling of the contractile cytoskeleton. Integrins sense load and have been implicated in cardiac hypertrophy, but how they discriminate between the two types of cardiac stress and translate mechanical loads into specific cytoskeletal signaling pathways is not clear. Here, we report that the focal adhesion protein β-parvin is highly expressed in cardiomyocytes and facilitates the formation of cell protrusions, the serial assembly of newly synthesized sarcomeres, and the hypertrophic growth of neonatal rat ventricular cardiomyocytes (NRVCs) invitro. In addition, physiological mechanical loading of NRVCs by either the application of cyclic, uni-axial stretch, or culture on physiologically stiff substrates promotes NRVC elongation in a β-parvin-dependent manner, which is achieved by binding of β-parvin to α/β-PIX, which in turn activates Rac1. Importantly, loss-of-function studies in mice also revealed that β-parvin is essential for the exercise-induced cardiac hypertrophy response invivo. Our results identify β-parvin as a novel mechano-responsive signaling hub in hypertrophic cardiomyocytes that drives cell elongation in response to physiological mechanical loads.

ARHGEF18: A new mechanosensitive player in endothelial cell biology?

Hemodynamic forces, particularly shear stress (SS), play a crucial role in endothelial cell (EC) physiology maintaining homeostasis of the vasculature. Alteration in SS or in its perception results in aberrant EC behavior and has been shown to be associated with vascular disorders such as atherosclerosis or intracranial aneurysm. Cytoskeleton remodeling is an essential step in EC response to these forces. Its organization is mostly under the control of Rho proteins, which activity is controlled by guanine nucleotide exchange factors (RhoGEFs). We aim at identifying the endothelial RhoGEFs involved in the defective adaptation to mechanical forces resulting in vascular disorders. ECs were exposed do different type of SS either physiological (16 dynes/cm 2 ) or pathological (3 and 36 dynes/cm 2 ). We used a 3′SRP analysis followed by qPCR and Pull down assay to identify mechanosensitive RhoGEF expression and activity. Functional analyses were conducted using RNAi-mediated knockdown approach combined with cellular and molecular biology in order to understand the role of the selected RhoGEF. We first identified ARHGEF18 as a mechanosensitive candidate. We showed that ARHGEF18 interacted specifically with RhoA in ECs. Interestingly, ARHGEF18 activity was down regulated by both pathological low and high SS compared to physiological one. Silencing of ARHGEF18 under static condition decreased EC adhesion and focal adhesion maturation, reduced EC migration but increased their ability to form a vascular network on Matrigel. ARHGEF18-deficient. Interestingly, when exposed to physiological SS, those ECs failed to elongate with the flow and had less focal adhesion sites. No change was observed on ECs proliferation. Additionally, intracellular signalling pathway analysis revealed that p38 phosphorylation was decreased in ARHGEF18-deficient ECs under physiological SS. Finally, preliminary results suggested that ARHGEF18-deficient ECs exposed to TNFα and SS had a decreased inflammatory phenotype with a loss of COX2 expression. Both the activity and the expression of ARHGEF18 in EC are regulated by SS. Future work in vitro will focus on the intracellular mechanism of action of ARHGEF18 on focal adhesion and inflammation. A mice model targeting ARHGEF18 in EC will allow us to understand further ARHGEF18 contribution vascular diseases.

1456-P: Molecular Architecture of AR Action in Insulin Secretion in Male Pancreatic ß-Cells

Male mice lacking the androgen receptor (AR) in islet β cells exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of GLP-1 in mouse and human islets. To gain information on DHT-stimulated signaling networks promoting GSIS, we combined functional proteomics with highly selective pharmacological inhibitors in male human islets, and islets from genetic mouse models. We found that DHT-stimulated GSIS in human islets involved a signaling cascade including focal adhesion kinases, the nonreceptor tyrosine kinase SRC, phosphoinositide 3-kinase, and the activation of mTOR complex 2. To define the early network of AR interacting proteins, we performed DHT-stimulated AR immunoprecipitation in INS1 832/3 cells followed by mass spectrometry-based proteomics. We will discuss the AR interactome including: 1) adenylate cyclase-modulating G proteins Gαs and Gαi, cAMP-dependent PKA, and the cAMP-dependent protein LASP1 regulating actin-based cytoskeletal activities; 2) Actin cytoskeleton remodeling proteins promoting actin depolymerization, and 3) Vesicular maturation including the lysosomal vacuolar-type ATPase acidifying insulin secretory granules and the prohormone convertase 2 involved in proinsulin processing. Notably, GLP-1 addition to DHT decreased AR interaction with all these proteins and promoted AR interaction with microtubule motor proteins promoting vesicular transport, proteins involved in exocytic vesicle docking to the plasma membrane, as well as small GTPase activating proteins and guanine nucleotide exchange factors involved in actin remodeling, as well as vesicle trafficking. Together these experiments provide novel insights into the architecture of AR action in enhancing GSIS. Disclosure P.Mota de sa: None. M.Qadir: None. M.Blandino-rosano: None. J.Fan: None. W.Xu: None. E.Bernal-mizrachi: None. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

Elasticity-associated rebinding rate of molecular bonds between soft elastic media

A quantitative understanding of how cells interact with their extracellular matrix via molecular bonds is fundamental for many important processes in cell biology and engineering. In these interactions, the deformability of cells and matrix are usually comparable with that of the bonds, making their rebinding events globally coupled with the deformation states of whole systems. Unfortunately, this important principle is not realized or adopted in most conventional theoretical models for analyzing cellular adhesions. In this study, we considered a new theoretical model of a cluster of ligand-receptor bonds between two soft elastic bodies, in which the rebinding rates of ligands to receptors are described, by considering the deformation of the overall system under the influence of bond distributions. On the basis of theory of continuum and statistical mechanics, we obtained an elasticity-associated rebinding rate of open bonds in a closed analytical form that highly depends on the binding states and distributions of all other bonds as well as on the overall deformation energy stored in the elastic bodies and all closed bonds. On the basis of this elasticity-associated rebinding rate and by performing Monte Carlo simulations, we uncovered new mechanisms underlying the adhesion stability of molecular-bond clusters associated with deformable elastic bodies. Moreover, we revealed that the rebinding processes of molecular bonds is not only dependent on interfacial separation but is related to overall energy. This newly proposed rebinding rate may substantially improve our understanding of how cells adapt to their microenvironments by adjusting their mechanical properties through cytoskeleton remodeling.

Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters.

Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell’s physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of β-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.

Dematin inhibits glioblastoma malignancy through RhoA-mediated CDKs downregulation and cytoskeleton remodeling

Glioblastoma multiforme (GBM) is well known as a highly aggressive brain tumor subtype. Here, we show that overexpression (OE) of dematin actin-binding protein (DMTN) inhibits GBM proliferation and invasion by affecting cell cycle regulation and actin remodeling, respectively. RT–qPCR, western blotting, and immunohistochemical (IHC) staining demonstrated a significant reduction in DMTN expression in gliomas, especially in high-grade gliomas (HGG) compared with normal brains, which correlates with worse survival in HGG patients. Functional studies revealed inhibitory effects of DMTN on tumor proliferation and migratory capacities. The attenuation in tumor proliferative ability upon DMTN OE was accompanied by RhoA suppression and CDK1, CDK2, CDK4, and cyclin D1 downregulation, while RhoA rescue restored the proliferative phenotype. Meanwhile, overexpression of DMTN produced profoundly disorganized stress fibers, which led to impaired tumor invasion. Furthermore, DMTN overexpression produced substantial suppression of tumor growth upon subcutaneous and intracranial implantation in mice, and this was accompanied by significantly reduced vinculin expression and Ki67 positivity. Taken together, these findings demonstrate the role of DMTN in regulating GBM cell proliferation, actin cytoskeleton, and cell morphology and identify DMTN as a vital tumor suppressor in GBM progression.