Cytoskeletal Activity Research Articles

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids in vitro.

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.

Identification of Mammalian Proteins That Collaborate with Type III Secretion System Function: Involvement of a Chemokine Receptor in Supporting Translocon Activity

ABSTRACTThe type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, including Yersinia pseudotuberculosis. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from a Ccr5−/− knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis.

Cell Volume Fluctuations in MDCK Monolayers

Cells moving collectively in tissues constitute a form of active matter, in which collective motion depends strongly on driven fluctuations at the single-cell scale. Fluctuations in cell area and number density are often seen in monolayers, yet their role in collective migration is not known. Here we study density fluctuations at the single- and multicell level, finding that single-cell volumes oscillate with a timescale of 4 h and an amplitude of 20%; the timescale and amplitude are found to depend on cytoskeletal activity. At the multicellular scale, density fluctuations violate the central limit theorem, highlighting the role of nonequilibrium driving forces in multicellular density fluctuations.

The interplay of cell-cell and cell-substrate adhesion in collective cell migration.

Collective cell migration often involves notable cell-cell and cell-substrate adhesions and highly coordinated motion of touching cells. We focus on the interplay between cell-substrate adhesion and cell-cell adhesion. We show that the loss of cell-surface contact does not significantly alter the dynamic pattern of protrusions and retractions of fast migrating amoeboid cells (Dictyostelium discoideum), but significantly changes their ability to adhere to other cells. Analysis of the dynamics of cell shapes reveals that cells that are adherent to a surface may coordinate their motion with neighbouring cells through protrusion waves that travel across cell-cell contacts. However, while shape waves exist if cells are detached from surfaces, they do not couple cell to cell. In addition, our investigation of actin polymerization indicates that loss of cell-surface adhesion changes actin polymerization at cell-cell contacts. To further investigate cell-cell/cell-substrate interactions, we used optical micromanipulation to form cell-substrate contact at controlled locations. We find that both cell-shape dynamics and cytoskeletal activity respond rapidly to the formation of cell-substrate contact.

COP1 Jointly Modulates Cytoskeletal Processes and Electrophysiological Responses Required for Stomatal Closure

Reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. Here, we investigate how environmental and hormonal signals cause these rearrangements and find that COP1, a RING-finger-type ubiquitin E3 ligase, is required for degradation of tubulin, likely by the 26S proteasome. This degradation is required for stomatal closing. In addition to regulating the cytoskeleton, we show that cop1 mutation impaired the activity of S-type anion channels, which are critical for stomatal closure. Thus, COP1 is revealed as a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function.

Protection after stroke: cellular effectors of neurovascular unit integrity.

Neurological disorders are prevalent worldwide. Cerebrovascular diseases (CVDs), which account for 55% of all neurological diseases, are the leading cause of permanent disability, cognitive and motor disorders and dementia. Stroke affects the function and structure of blood-brain barrier, the loss of cerebral blood flow regulation, oxidative stress, inflammation and the loss of neural connections. Currently, no gold standard treatments are available outside the acute therapeutic window to improve outcome in stroke patients. Some promising candidate targets have been identified for the improvement of long-term recovery after stroke, such as Rho GTPases, cell adhesion proteins, kinases, and phosphatases. Previous studies by our lab indicated that Rho GTPases (Rac and RhoA) are involved in both tissue damage and survival, as these proteins are essential for the morphology and movement of neurons, astrocytes and endothelial cells, thus playing a critical role in the balance between cell survival and death. Treatment with a pharmacological inhibitor of RhoA/ROCK blocks the activation of the neurodegeneration cascade. In addition, Rac and synaptic adhesion proteins (p120 catenin and N-catenin) play critical roles in protection against cerebral infarction and in recovery by supporting the neurovascular unit and cytoskeletal remodeling activity to maintain the integrity of the brain parenchyma. Interestingly, neuroprotective agents, such as atorvastatin, and CDK5 silencing after cerebral ischemia and in a glutamate-induced excitotoxicity model may act on the same cellular effectors to recover neurovascular unit integrity. Therefore, future efforts must focus on individually targeting the structural and functional roles of each effector of neurovascular unit and the interactions in neural and non-neural cells in the post-ischemic brain and address how to promote the recovery or prevent the loss of homeostasis in the short, medium and long term.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

Efferocytosis ő autophagy crosstalk in alveolar macrophages exposed to cigarette smoking (151.7)

Alveolar macrophages (AM) apoptotic cell clearance function (efferocytosis) is dysregulated in COPD due to cigarette smoke (CS) exposure via production of ceramides and sphingosine (SHP). CS also alters the maturation of the autophagosomes (AP) blocking the autophagic flux. We hypothesized that impairment of both AP maturation and efferocytosis share an identical mechanism of cytoskeletal derangement linked to HDAC6 inactivation and sphingosine accumulation. AM obtained via bronchoalveolar lavage from human smoker and non‐smoker donors or from Sprague Dawley rats exposed to CS (3 months) were subject to engulfment assays quantified by flow cytometry. HDAC6 was evaluated by real‐time PCR or Western blot. Both CS and SPH significantly impaired efferocytosis in rat and human AM (50% and 80% inhibition; p<0.05, respectively). HDAC6 protein activity was markedly impaired by CS exposure. This effect was in part mediated by SPH production, since a ceramidase inhibitor, MAPP, restored both HDAC6 levels and improved autophagic flux. Moreover, HDAC6 inhibition with BML‐281 or siRNA significantly impaired efferocytosis, as well as bafilomycin treatment, an inhibitor of AP maturation (by up to 7‐fold; p<0.05). Whole protein HDAC6 overexpression significantly restored the efferocytosis impairment induced by CS but overexpression of the HDAC6 catalytic mutant did not. These results indicate a novel role of HDAC6 in efferocytosis. The impairment of both autophagic flux and efferocytosis may be mechanistically linked at the level of sphingolipid regulation of cytoskeletal effectors activity such as HDAC6. Improvement in AP maturation may help restore efferocytosis, an essential step in the resolution of pulmonary inflammation.Grant Funding Source: ALA RG‐1966666‐N and NIH‐HL077328

Restless cell syndrome

Restless cell syndrome

Cytoskeletal Stiffness Controls the Threshold of T Cell Activation

T cells utilize a dynamic cytoskeleton to effectively scan antigen-presenting cells and become fully activated. Cytoskeletal activities help the T cell both generate mechanical forces and receive these forces from extracellular matrix and from other cells. To study the function of cytoskeletal changes during T cell activation, we modified an atomic force microscope (AFM) to deliver antigen tethered to the AFM cantilever, simultaneously ligating T-cell receptors (TCRs) and measuring changes in cytoskeletal elasticity (stiffness). We found that T cells undergo rapid cytoskeletal changes upon TCR triggering. Because traditional AFM techniques preclude measuring these cytoskeletal changes in real time, we developed a new high-bandwidth cantilever that boosts scanning speeds by 1000-fold. We show that the cytomechanical stiffness actually regulates the threshold of activation of T cells. Our results highlight the functional importance of the cytoskeleton in tuning cellular responses to receptor signals and will be of broad interest to all cell biologists interested in cellular mechanotransduction.

A Proteomic Profile of Cervical Remodeling in Mice during Early and Late Pregnancy

Objective: Decipher mechanisms that underlie cervical remodeling (CR) during pregnancy. Design and population: Examine the proteomic profile of mice cervices in early and late pregnancy and verify data generated using Western Blot and real time PCR analysis. Main outcome measures: Identify signature proteins. Results: In total, seventy three (73) signature proteins were identified and were found up regulated in pregnancy, namely proteins associated with: a) collagen presence and organization (4), notably biglycan and lumican, b) immune scavenging functions and c) cytoskeletal activity. These proteomic trends were verified through Western blot and real time PCR analysis. Conclusion: These signature proteins and their expression profile collectively suggest a complex biological process involving extracellular matrix (ECM)- and immune`-associated factors. Based on these data, we propose that biglycan may provide the link between natural birth and inflammation-mediated pre-term birth.

Active cytoskeletal force and chromatin condensation independently modulate intranuclear network fluctuations

Chromatin remodeling, including the movement of genes and regulatory factors, precedes or accompanies stimulated changes in gene expression. Here we quantify chromatin fluctuations in primary human cells using particle-tracking microrheology and determine the physical mechanisms which influence chromatin reorganization. We find that intranuclear movements are enhanced beyond thermal motion by active force generation from cytoskeletal motor activity propagated through the LINC complex; intranuclear movements are also dependent on the viscoelasticity of the DNA-protein polymer network. Chromatin movements were dramatically altered by modulation of chromatin condensation state, which we independently verified using fluorescence lifetime imaging microscopy (FLIM). These findings suggest that chromatin condensation and cytoskeletal force generation play distinct functional roles in regulating intranuclear movements, and these effects are decoupled as measured by particle tracking. We further utilize this approach in identifying the nuclear responsiveness of primary human endothelial cells to vascular endothelial growth factor (VEGF): early in the response chromatin movements increase and are dominated by cytoskeletal force, which transitions at later times to a chromatin decondensation event. Given the hierarchical genome organization in primary cells, our work generally suggests an important role for force generation and chromatin mechanics in altered gene expression kinetics.

Abrogation of Chronic Rejection in Rat Model System Involves Modulation of the mTORC1 and mTORC2 Pathways

Current immunosuppressive regimens fail to avert chronic rejection (CR) of transplanted organs; however, selective targeting of actin-cytoskeletal regulators decreases T-cell motility and abrogates CR in rat model system. Administration of mutated class I major histocompatibility complex molecules or selective targeting of the RhoA pathway, which controls T-cell cytoskeletal activity, using Y27632 (a selective Rock1 inhibitor) resulted in reduced T-cell infiltration and abrogation of CR as judged from the neointimal index (13.9±19.7 vs. 45±37.5; P<0.001) and the number of affected vessels (30% vs. 60%; P<0.01). Here, we examined the role of mammalian target of rapamycin (mTOR) pathway in inhibition of CR. A mutated class I major histocompatibility complex molecule that eliminates CR was delivered into ACI recipients of Wistar-Furth hearts at the time of transplantation with subtherapeutic cyclosporine (10 mg/kg on days 0-2). Controls included untreated and cyclosporine A-treated (10 mg/kg on days 0-2) heart allograft recipients. Western blotting and immunostaining showed that rat heart allografts with abolished CR exhibited down-regulation of the RAPA-sensitive mTORC1 components such as mTOR and Raptor and down-regulation of the RAPA-insensitive mTORC2 elements Rictor and Sin1. The mTOR regulator Deptor and its downstream target Rac1 were also inhibited. Abrogation of CR in rat model system involves modulation of two mTOR pathways: a RAPA-sensitive mTORC1 pathway regulating cellular proliferation and a RAPA-insensitive mTORC2 pathway regulating T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies.

An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration

It is generally believed that cytoskeletal activities drive random cell migration while signal transduction events initiated by receptors regulate the cytoskeleton to guide cells. However, we find that the cytoskeletal network, involving Scar/Wave, Arp 2/3, and actin binding proteins, is only capable of generating rapid oscillations and undulations of the cell boundary. The signal transduction network, comprising multiple pathways that include Ras GTPases, PI3K, and Rac GTPases, is required to generate the sustained protrusions of migrating cells. The signal transduction network is excitable, displaying wave propagation, refractoriness, and maximal response to suprathreshold stimuli, even in the absence of the cytoskeleton. We suggest that cell motility results from coupling of “pacemaker” signal transduction and “idling motor” cytoskeletal networks, and various guidance cues that modulate the threshold for triggering signal transduction events are integrated to control the mode and direction of migration.

Interplay of cytoskeletal activity and lipid phase stability in dynamic protein recruitment and clustering

Recent experiments have revealed that some membrane proteins aggregate to form clusters. This type of process has been proven to be dynamic and to be actively maintained by external kinetics. Additionally, this dynamic recruiting is cholesterol- and actin-dependent, suggesting that raft organization and cytoskeleton rearrangement play a crucial role. In the present study, we propose a simple model that provides a general framework to describe the dynamical behavior of lipid-protein assemblies. Our results suggest that lipid-mediated interactions and cytoskeleton-anchored proteins contribute to the modulation of such behavior. In particular, we find a resonant condition between the membrane protein and cytoskeleton dynamics that results in the invariance of the ratio of clustered proteins that is found in in vivo experimental observations.

The actin-microtubule cross-linking activity ofDrosophilaShort stop is regulated by intramolecular inhibition

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis--much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a "closed" conformation through interactions between its NH(2)-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH(2)-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.

Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear

Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott–Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.

Epigallocatechin-3 gallate inhibits cancer invasion by repressing functional invadopodia formation in oral squamous cell carcinoma

Although the polyphenol EGCG has various beneficial biological effects, its effect on cytoskeletal activities during cancer invasion is not well defined, and the precise molecular mechanisms are largely unknown. Here, we provide molecular evidence on the anti-invasion effect of EGCG in OSCC cells using an in vitro 3-D culture system and in vivo athymic mouse model. Briefly, EGCG exerted an inhibitory effect on the Matrigel-based Transwell invasion and migration of OSCC cells. These effects were not due to decreased cell viability or adhesion capacity to ECM. EGCG-treated OSCC cells possessed fully extended actin fibers without invadopodia, indicating a loss of ECM degradation capacity. Decreased phosphorylation of Src, CTTN, and FAK also followed EGCG treatment. Additionally, EGCG reduced activation of RhoA in dominant-negative RhoA N19 and constitutively active RhoA Q63E cells, and inhibited the invasive capability of these cells in the 3-D cell growth model. Furthermore, the administration of EGCG led to substantial inhibition of tumor growth and activation of invadopodial proteins in the tumor tissues of mice inoculated with OSCC cells. The data indicate the potential value of EGCG as an invadopodia-targeted anti-invasive agent in cancer therapy.

Traffic of Secondary Metabolites to Cell Surface in the Red Alga Laurencia dendroidea Depends on a Two-Step Transport by the Cytoskeleton

In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the chemical defence of red algae.

PP117: Does podoplanin contribute to the cytoskeletal remodelling activity in ameloblastomas?

Purpose To investigate the relationship between podoplanin expression and cytoskeletal remodeling activity of the odontogenic epithelium of ameloblastomas. Material and methods Nineteen paraffin-embedded ameloblastomas were immunohistochemically analyzed using anti-human podoplanin and anti-human ezrin antibodies. The membranous and cytoplasmic expression of both antibodies in the neoplastic odontogenic epithelial cells was evaluated using a scoring method. The correlation between the immunostaining of ezrin and podoplanin in ameloblastomas was verified through the Spearman’s correlation test and membranous expression was compared with cytoplasmic expression of both antibodies through Wilcoxon test. The level of significance was set at 5% for all tests. Results All ameloblastomas displayed podoplanin and ezrin expression in ameloblast-like cells of the epithelial islands and cords. The majority (94.7%) of the neoplasias presented high expression of ezrin. Cytoplasmic expression of ezrin was statistically stronger than membranous staining of this protein in the tumors ( p = 0.025). Membranous expression of podoplanin was inversely correlated with membranous ezrin expression ( r = −0.66; p = 0.002). Conclusion These results suggest a synergistic role of cytoplasmic ezrin and membranous podoplanin expressions in the local invasion process of ameloblastomas.