Actin Membrane Research Articles

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA–mDia2 signaling and filopodium formation

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with β1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin–binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA–mDia2–regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

Protective Effects of Carbon Monoxide Delivered By Corm-401 in Hyperhemolysis in Patients with Sickle Cell Disease

IntroductionHyperhemolysis has been well described in delayed hemolytic transfusion reaction (DHTR), which is one of the most serious complications of transfusion especially in patients with sickle cell disease (SCD). In the most severe cases, this life-threatening syndrome may reach 10% lysis of total red blood cells (RBCs) (Habibi A et. al. Am J Hematol 2016). DHTR is characterized by an acute anemia with increased plasma free Hb and free heme. Hyperhemolysis can also be found in other hemoglobinopathies and extracorporeal circulation procedures.Most in vitro studies on hyperhemolysis used only purified Hb or heme which did not allow to investigate all deleterious effects on the endothelium. We have developed a global and physiopathologic approach to assess the mechanism of hyperhemolysis-induced endothelial dysfunction. For this purpose, we created a microfluidic model reproducing hyperhemolysis observed in the most severe DHTR but it's also applicable for other pathogenesis of hyperhemolysis.Among the potential therapeutic approaches, carbon monoxide (CO) is beneficial on endothelial cells by its anti-inflammatory, antioxidant and vasodilator effect which reduce the toxicity of Hb and improve tissue perfusion (Motterlini R et.al. Nat. Rev. Drug Discov 2010). Here we investigated the therapeutic effects of a CO-releasing molecule, CORM-401, in hyperhemolysis-induced endothelial dysfunction.Materials and methodsFlow culture Human Umbilical Vein Endothelial Cells (HUVECs) in fibronectin-coated µ-Slides were preconditioned for 4 hours under shear stress of 1dyn/cm² with sonicated RBCs of healthy donors (AA) reconstituted in serum of either AA or SCD patients (SS) at 7 g/L of free Hb. In negative control conditions, cells were pretreated similarly with either AA serum or culture medium. Cells were then collected for analyses of actin network, membrane markers and endothelial function. Treatment with CORM-401 or inactive CORM (iCORM) was performed at fixed concentration during the preconditioning and perfusion steps.ResultsHemolysis induces multiple endothelial damage and dysfunctionAfter exposure to hemolysate, we noted a significant increase of subendothelial matrix exposition (fibronectin-coated surfaces) due to actin network reorganization and cells detachment. Immunofluorescence staining showed a moderate activation of HUVECs induced by hemolysate with membrane expression of adhesion molecules such as ICAM-1, E-Selectin and VCAM-1, but not VWF and P-Selectin. At a similar free Hb level, preconditioning with hemolysate without microparticles (MPs) had the lowest deleterious impact on HUVECs suggesting a role of MPs in hyperhemolysis.Perfusion of AA whole blood on HUVECs pretreated with hemolysate resulted in aggregation and activation of platelets in a GPIIbIIIa-dependent manner at injury sites. We also observed a significant increase of adhered RBCs (14-fold compared to control).Compared to whole blood RBC lysis, purified Hb induced similar subendothelial exposure and RBC adherence but did not lead to significant HUVECs activation.To study the DHTR in RBC transfusion of SCD patients, SS serum was used to perform hemolysis preconditioning of HUVECs. Adherence of RBCs was similar to condition using AA serum suggesting that endothelial damage in DHTR of SCD patients depend on hemolysate compositions and RBC MPs, rather than on patients' serum.Effects of CORM-401 on hemolysis-induced endothelial dysfunctionAdding CORM-401 (50 to 100 µM) to the hemolysate produced 15% free COHb leading to a concentration-dependent decrease in hemolysis-induced HUVECs activation and RBC adherence (Figure 1 C, E). While no significant effects at the subendothelial exposure were observed, the aggregation of platelets at injury sites was decreased after CORM-401 treatment compared to controls (Figure 1 A, B, D), confirming the anti-platelet aggregation effect of this molecule (Chlopicki S et. al. Naunyn Schmiedebergs Arch Pharmacol 2012).ConclusionOur in vitro model enabled the reproduction of endothelial damage by hyperhemolysis and to determine the deleterious effects of each hemolysate component. The in vitro beneficial effects of CORM-401 were also demonstrated and an in vivo study on SCD mice is underway to explore the therapeutic effects of this molecule against hyperhemolysis-induced endothelial damage. [Display omitted] DisclosuresBartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

Nanoscale Topography–Rigidity Correlation at the Surface of T Cells

The mechanical properties of cells affect their function, in sensing, development, and motility. However, the rigidity of the cell surface and its correlation to its local topography remain poorly understood. Here, we applied quantitative imaging AFM to capture high-resolution force maps at the surface of nonadherent T cells. Using this method, we found a positive topography-rigidity correlation at the cells' surface, as opposed to a negative correlation at the surface of adherent cells. We then used 3D single-molecule localization microscopy of the membrane and cortical actin and an actin-perturbing drug to implicate actin involvement in the positive rigidity-topography correlation in T cells. Our results clearly reveal the variability of cell-surface rigidity and its underlying mechanism, showing a functional role for cortical actin in the PM protrusions of T cells, since they are locally more rigid than their surroundings. These findings suggest the possible functional role of membrane protrusions as mechanosensors.

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga, Silvetia babingtonii (Fucales, Phaeophyceae)

ABSTRACTIn the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.

ADP-ribosylation factor-like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation.

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)–like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394–1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

3pM_K3Non-invasive Live-imaging of Plasma Membrane and Cortical Actin Dynamics in Endocytic Process by High-speed Atomic Force Microscopy

3pM_K3Non-invasive Live-imaging of Plasma Membrane and Cortical Actin Dynamics in Endocytic Process by High-speed Atomic Force Microscopy

Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways.

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase (CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter (SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin (ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin (DSP ↑2.4 MFM), and basement membrane usherin (USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle.

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration, as well as the caveolae-associated protein cavin-1, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration, as well as the caveolae-associated protein cavin-1, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes

SummaryHow cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kβ across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion.

Effects of nanosilver on Mytilus galloprovincialis hemocytes and early embryo development

Silver nanoparticles (AgNP), one of the main nanomaterials for production and use, are expected to reach the aquatic environment, representing a potential threat to aquatic organisms. In this study, the effects of bare AgNPs (47 nm) on the marine mussel Mytilus galloprovincialis were evaluated at the cellular and whole organism level utilizing both immune cells (hemocytes) and developing embryos. The effects were compared with those of ionic Ag+(AgNO3). In vitro short-term exposure (30 min) of hemocytes to AgNPs induced small lysosomal membrane destabilization (LMS EC50 = 273.1 μg/mL) and did not affect other immune parameters (phagocytosis and ROS production). Responses were little affected by hemolymph serum (HS) as exposure medium in comparison to ASW. However, AgNPs significantly affected mitochondrial membrane potential and actin cytoskeleton at lower concentrations. AgNO3 showed much higher toxicity, with an EC50 = 1.23 μg/mL for LMS, decreased phagocytosis and induced mitochondrial and cytoskeletal damage at similar concentrations.Both AgNPs and AgNO3 significantly affected Mytilus embryo development, with EC50 = 23.7 and 1 μg/L, respectively. AgNPs caused malformations and developmental delay, but no mortality, whereas AgNO3 mainly induced shell malformations followed by developmental arrest or death.Overall, the results indicate little toxicity of AgNPs compared with AgNO3; moreover, the mechanisms of action of AgNP appeared to be distinct from those of Ag+. The results indicate little contribution of released Ag+ in our experimental conditions. These data provide a further insight into potential impact of AgNPs in marine invertebrates.

Interplay between membrane tension and the actin cytoskeleton determines shape changes

The ability of mammalian cells to deform their membrane relies on the action of the cytoskeleton. In particular, the dynamics of the actin cytoskeleton, assembling at the plasma membrane, plays a crucial role in controlling cell shape. Many proteins are involved to ensure proper growth of the actin network at the cell membrane. The detailed structure of this network regulates the force that is necessary for membrane deformation. We address here how the presence of capping proteins, which limit the length of actin filaments and thus affects network topology, influences membrane shape. We use a system of liposomes, activated to polymerize actin at their surface, and placed in a mixture of purified proteins that reconstitutes actin dynamics. Our system also allows the variation of membrane tension by deflating the liposomes. We show that membrane deformations are clearly favored in the presence of capping proteins in the actin network. Moreover, in the absence of capping proteins, membrane deformations appear only when the liposomes are deflated. Our results unveil that the interplay between membrane tension and actin network structure and dynamics governs cell shape.

PO-286 Protein kinase D3: a crucial regulator of breast cancer stem cell survival

IntroductionTriple negative breast cancer (TNBC) accounts for 15%–20% of all breast carcinomas in women. TNBC is particularly aggressive, highly metastatic and associated with poor prognosis. We previously identified Protein Kinase D3 (PKD3) to be upregulated in TNBC. PKD3 is a serine-threonine kinase that is best known for its functions in membrane trafficking and actin remodelling. Here we identify a novel role for PKD3 in breast cancer stem cell (CSC) maintenance. CSCs are endowed with tumor-initiating capacity, they possess increased chemoresistance and are known to initiate metastasis, and therefore are responsible for tumour relapse.Material and methodsIn the TNBC cell line MDA-MB-231 with a stable PKD3 knockdown, we performed a flow cytometry based screen of cell surface proteins, revealing the downregulation of several membrane proteins associated with CSCs. The importance of PKD3 for driving a CSC gene expression signature, maintaining CSC numbers and function was tested by qRT-PCR analysis of CSC markers, by measuring aldehyde dehydrogenase (ALDH) activity and performing mammosphere assays, respectively. Paclitaxel sensitivity of TNBC cell lines was assessed in combination with the selective PKD inhibitor CRT0066101. Whether the ectopic expression of PKD3 was sufficient to induce the expansion of the CSC pool was analysed in the non-transformed breast epithelial cell line MCF10A.Results and discussionsIn MDA-MB-231 cells with PKD3 knockdown, the CSC markers (CD184, Notch4, Sox2, Oct3/4) were downregulated. This was associated with reduced CSC numbers based on ALDH positivity and sphere-forming efficiency in mammosphere assays. Pharmacological PKD inhibition in the TNBC cell lines MDA-MB-231, BT549 and MDA-MB-436 verified these results. By contrast, inducible PKD3 expression in MCF10A cells elevated mammosphere formation and stem cell marker expression. Importantly, the combination of paclitaxel with CRT0066101 was superior over the single treatments in suppressing the survival of MDA-MB-231, BT549 and MDA-MB-436 cells in both colony formation and mammosphere assays.ConclusionOur results establish PKD3 as a crucial regulator of CSC maintenance and demonstrate that paclitaxel treatment in combination with PKD inhibition provides a potential novel strategy for future therapeutic applications in TNBC treatment.

Chemical crosslinking analysis of β-dystroglycan in dystrophin-deficient skeletal muscle

Background: In Duchenne muscular dystrophy, primary abnormalities in the membrane cytoskeletal protein dystrophin trigger the loss of sarcolemmal linkage between the extracellular matrix component laminin-211 and the intracellular cortical actin membrane cytoskeleton. The disintegration of the dystrophin-associated glycoprotein complex renders the plasma membrane of contractile fibres more susceptible to micro-rupturing, which is associated with abnormal calcium handling and impaired cellular signalling in dystrophinopathy. Methods: The oligomerisation pattern of β-dystroglycan, an integral membrane protein belonging to the core dystrophin complex, was studied using immunoprecipitation and chemical crosslinking analysis. A homo-bifunctional and non-cleavable agent with water-soluble and amine-reactive properties was employed to study protein oligomerisation in normal versus dystrophin-deficient skeletal muscles. Crosslinker-induced protein oligomerisation was determined by a combination of gel-shift analysis and immunoblotting. Results: Although proteomics was successfully applied for the identification of dystroglycan as a key component of the dystrophin-associated glycoprotein complex in the muscle membrane fraction, mass spectrometric analysis did not efficiently recognize this relatively low-abundance protein after immunoprecipitation or chemical crosslinking. As an alternative approach, comparative immunoblotting was used to evaluate the effects of chemical crosslinking. Antibody decoration of the crosslinked microsomal protein fraction from wild type versus the mdx-4cv mouse model of dystrophinopathy revealed oligomers that contain β-dystroglycan. The protein exhibited a comparable reduction in gel electrophoretic mobility in both normal and dystrophic samples. The membrane repair proteins dysferlin and myoferlin, which are essential components of fibre regeneration and counteract the dystrophic phenotype, were also shown to exist in high-molecular mass complexes. Conclusions: The muscular dystrophy-related reduction in the concentration of β-dystroglycan, which forms in conjunction with its extracellular binding partner α-dystroglycan a critical plasmalemmal receptor for laminin-211, does not appear to alter its oligomeric status. Thus, independent of direct interactions with dystrophin, this sarcolemmal glycoprotein appears to exist in a supramolecular assembly in muscle.

Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes.

Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90's relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90's effect on pig oocyte meiotic maturation. Endogenous Hsp90α was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90α protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90α by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90α can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes.

Phospho-regulation of intrinsically disordered proteins for actin assembly and endocytosis.

Actin filament assembly contributes to the endocytic pathway pleiotropically, with active roles in clathrin-dependent and clathrin-independent endocytosis as well as subsequent endosomal trafficking. Endocytosis comprises a series of dynamic events, including the initiation of membrane curvature, bud invagination, vesicle abscission and subsequent vesicular transport. The ultimate success of endocytosis requires the coordinated activities of proteins that trigger actin polymerization, recruit actin-binding proteins (ABPs) and organize endocytic proteins (EPs) that promote membrane curvature through molecular crowding or scaffolding mechanisms. A particularly interesting phenomenon is that multiple EPs and ABPs contain a substantial percentage of intrinsically disordered regions (IDRs), which can contribute to protein coacervation and phase separation. In addition, intrinsically disordered proteins (IDPs) frequently contain sites for post-translational modifications (PTMs) such as phosphorylation, and these modifications exhibit a high preference for IDR residues [Groban ES et al. (2006) PLoS Comput Biol 2, e32]. PTMs are implicated in regulating protein function by modulating the protein conformation, protein-protein interactions and the transition between order and disorder states of IDPs. The molecular mechanisms by which IDRs of ABPs and EPs fine-tune actin assembly and endocytosis remain mostly unexplored and elusive. In this review, we analyze protein sequences of budding yeast EPs and ABPs, and discuss the potential underlying mechanisms for regulating endocytosis and actin assembly through the emerging concept of IDR-mediated protein multivalency, coacervation, and phase transition, with an emphasis on the phospho-regulation of IDRs. Finally, we summarize the current understanding of how these mechanisms coordinate actin cytoskeleton assembly and membrane curvature formation during endocytosis in budding yeast.

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both invitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.

Mammalian nonmuscle myosin II comes in three flavors

Nonmuscle myosin II is an actin-based motor that executes numerous mechanical tasks in cells including spatiotemporal organization of the actin cytoskeleton, adhesion, migration, cytokinesis, tissue remodeling, and membrane trafficking. Nonmuscle myosin II is ubiquitously expressed in mammalian cells as a tissue-specific combination of three paralogs. Recent studies reveal novel specific aspects of their kinetics, intracellular regulation and functions. On the other hand, the three paralogs also can copolymerize and cooperate in cells. Here we review the recent advances from the prospective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cell.

Localization of EFA6 (exchange factor for ARF6) isoform D in steroidogenic testicular Leydig cells of adult mice

EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP and the resulting activated form of Arf6 is involved in the membrane trafficking and actin remodeling of cells. Our previous study has shown the selective expression/localization of EFA6D in steroidogenic adrenocortical cells in situ of adult mice. In view of the previous finding, the present study was undertaken to examine its localization in mouse Leydig cells representing another steroidogenic cell species in order to further support the possible involvement of the EFA6/Arf6 cascade via membrane trafficking in the regulation of steroidogenesis and/or secretion. A distinct band for EFA6D with the same size as that of the brain was detected in the testis of adult mice. In immuno-light microscopy, immunoreactivity for EFA6D was seen throughout the cytoplasm in most Leydig cells without any distinct accumulation along the plasmalemma. Lack of immunoreactivity for EFA6D was seen in the seminiferous tubular epithelium. In immuno-electron microscopy, the immune-labeling was seen in sporadic/focal patterns on plasma membranes and some vesicles and vacuoles subjacent to the plasma membranes. More constant and rather predominant is the labeling on numerous mitochondria. No immuno-labeling was seen in lipid droplets. The present study suggests that EFA6D is somehow involved in regulation of the synthesis and/or secretion of testosterone through the membrane-traffic by activation of Arf6. In addition, EFA6D is suggested to play in mitochondria some yet unidentified roles rather independent of Arf6-activation, which remains to be elucidated.