Actin Depolymerization Research Articles

Mo1568 - Integrated Multi-Omic Analysis Reveals Female Predominance of Deregulated Mucosal Actin Depolymerization by Decreased Tesk1-Mediated CFL1-Phosphorylation in IBS-D

Mo1568 - Integrated Multi-Omic Analysis Reveals Female Predominance of Deregulated Mucosal Actin Depolymerization by Decreased Tesk1-Mediated CFL1-Phosphorylation in IBS-D

Downregulation of LIMK1\u2013ADF/cofilin by DADS inhibits the migration and invasion of colon cancer

This study aimed to explore whether the downregulation of LIM kinase 1 (LIMK1)-actin depolymerization factor (ADF, also known as destrin)/cofilin by diallyl disulfide (DADS) inhibited the migration and invasion of colon cancer. Previous studies have shown that silencing LIMK1 could significantly enhance the inhibitory effect of DADS on colon cancer cell migration and invasion, suggesting that LIMK1 was a target molecule of DADS, which needed further confirmation. This study reported that LIMK1 and destrin were highly expressed in colon cancer and associated with poor prognosis of patients with colon cancer. Also, the expression of LIMK1 was positively correlated with the expression of destrin. The overexpression of LIMK1 significantly promoted colon cancer cell migration and invasion. DADS obviously inhibited migration and invasion by suppressing the phosphorylation of ADF/cofilin via downregulation of LIMK1 in colon cancer cells. Furthermore, DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1 in vitro and in vivo, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicated that LIMK1 was a potential target molecule for the inhibitory effect of DADS on colon cancer cell migration and invasion.

Single-Particle Tracking Reveals a Dynamic Role of Actin Filaments in Assisting Long-Range Axonal Transport in Neurons

Abstract Here, we demonstrated that actin filaments mediate axonal transport in dorsal root ganglia (DRG) neurons using fluorescence single-particle tracking. We employed a compartmentalized microfluidic cell culturing chamber that allows depolymerization of actin filaments within an axonal segment. We observed that local actin depolymerization results in a two-fold increase in the average pausing duration, whereas the microtubule-dependent instantaneous transport speed is not perturbed. Collectively, our data reveal an important role of actin filaments in assisting microtubule-dependent long-range NGF axonal transport in DRG neurons.

Cofilin 1 induces the epithelial-mesenchymal transition of gastric cancer cells by promoting cytoskeletal rearrangement.

Epithelial-mesenchymal transition (EMT) is an important biological process whereby malignant tumor cells obtain the ability to migrate, invade, resist apoptosis and degrade the extracellular matrix. We found that Cofilin1 (CFL1) expression was elevated in clinical gastric cancer specimens and correlated with biomarkers of EMT in BGC-823 gastric cancer cells. BGC-823 cells exhibited EMT phenotypes and increased metastatic ability when induced by TGF-β1. By contrast, BGC-823 cells transfected with Lv-siRNA-CFL1 did not exhibit EMT phenotypes under the same inducing conditions. As CFL1 expression increased, EMT cell filopodia stretched out. In addition, the ultrastructures observed using transmission electron microscopy indicated that silencing of CFL1 markedly inhibited depolymerization of fibrous actin and cytoskeletal reorganization during EMT. Similar results were obtained in vivo. These findings demonstrate that CFL1 induces EMT by promoting cytoskeletal rearrangement. Our results may provide the basis for developing new anticancer drugs to inhibit CFL1.

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway.

Rosiglitazone drives cavin-2/SDPR expression in adipocytes in a CEBPα-dependent manner.

Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.

Identification of sennoside A as a novel inhibitor of the slingshot (SSH) family proteins related to cancer metastasis

Phospho-cofilin (p-cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John’s wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function

SummaryThe active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

Oxidation of F-actin controls the terminal steps of cytokinesis

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division.

Actin visualization at a glance

Actin functions in a multitude of cellular processes owing to its ability to polymerize into filaments, which can be further organized into higher-order structures by an array of actin-binding and regulatory proteins. Therefore, research on actin and actin-related functions relies on the visualization of actin structures without interfering with the cycles of actin polymerization and depolymerization that underlie cellular actin dynamics. In this Cell Science at a Glance and the accompanying poster, we briefly evaluate the different techniques and approaches currently applied to analyze and visualize cellular actin structures, including in the nuclear compartment. Referring to the gold standard F-actin marker phalloidin to stain actin in fixed samples and tissues, we highlight methods for visualization of actin in living cells, which mostly apply the principle of genetically fusing fluorescent proteins to different actin-binding domains, such as LifeAct, utrophin and F-tractin, as well as anti-actin-nanobody technology. In addition, the compound SiR-actin and the expression of GFP-actin are also applicable for various types of live-cell analyses. Overall, the visualization of actin within a physiological context requires a careful choice of method, as well as a tight control of the amount or the expression level of a given detection probe in order to minimize its influence on endogenous actin dynamics.

Microfilament Depolymerization Is a Pre-requisite for Stem Cell Formation During In vitro Shoot Regeneration in Arabidopsis.

De novo shoot regeneration is widely used in fundamental studies and agricultural applications. Actin microfilaments are involved in many aspects of plant cell division, cell morphogenesis and cell signal transduction. However, the function of actin microfilaments during de novo shoot regeneration is poorly understood. Here, we investigated the organization of actin microfilaments during this process and found that stem cell formation was associated with microfilament depolymerization. Furthermore, inhibition of microfilament depolymerization by phalloidin treatment or downregulation of actin depolymerizing factors (ADFs) restrained stem cell initiation and shoot regeneration. Inhibition of ADF expression affected the architecture of microfilaments during stem cell formation, and the polar transport and distribution of auxin were also disrupted. Together, our results demonstrate that organization of the microfilament cytoskeleton play important roles in stem cell formation and shoot meristem induction during shoot regeneration.

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4-dichlorophenoxyacetic acid-mediated alteration of actin in Arabidopsis roots.

2,4-Dichlorophenoxyacetic acid (2,4-D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole-3-acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4-D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4-D-specific mutants suggested that 2,4-D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4-D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4-D but not IAA altered the actin structure in long-term and short-term assays. Analysis of the 2,4-D-specific mutant aar1-1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4-D-induced depolymerization of actin. The ubiquitin proteasome mutants tir1-1 and axr1-12, which show enhanced resistance to 2,4-D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4-D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4-D on the organization of actin filaments. Roots of the double mutant aar1-1 tir1-1 also showed enhanced resistance to 2,4-D-induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4-D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCFTIR1 ubiquitin proteasome components.

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-βH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.

Bone marrow transplantation improves proximal tubule dysfunction in a mouse model of Dent disease

Dent disease is a rare X-linked tubulopathy caused by mutations in the endosomal chloride-proton exchanger (ClC-5) resulting in defective receptor-mediated endocytosis and severe proximal tubule dysfunction. Bone marrow transplantation has recently been shown to preserve kidney function in cystinosis, a lysosomal storage disease causing proximal tubule dysfunction. Here we test the effects of bone marrow transplantation in Clcn5Y/- mice, a faithful model for Dent disease. Transplantation of wild-type bone marrow in Clcn5Y/- mice significantly improved proximal tubule dysfunction, with decreased low-molecular-weight proteinuria, glycosuria, calciuria, and polyuria four months after transplantation, compared to Clcn5Y/- mice transplanted with ClC-5 knockout bone marrow. Bone marrow-derived cells engrafted in the interstitium, surrounding proximal tubule cells, which showed a rescue of the apical expression of ClC-5 and megalin receptors. The improvement of proximal tubule dysfunction correlated with Clcn5 gene expression in kidneys of mice transplanted with wild-type bone marrow cells. Coculture of Clcn5Y/- proximal tubule cells with bone marrow-derived cells confirmed rescue of ClC-5 and megalin, resulting in improved endocytosis. Nanotubular extensions between the engrafted bone marrow-derived cells and proximal tubule cells were observed invivo andinvitro. No rescue was found when the formation of the tunneling nanotubes was prevented by actin depolymerization or when cells were physically separated by transwell inserts. Thus, bone marrow transplantation may rescue the epithelial phenotype due to an inherited endosomal defect. Direct contacts between bone marrow-derived cells and diseased tubular cells play a key role in the rescue mechanism.

From the Cover: Zinc oxide Nanoparticles-Induced Reactive Oxygen Species Promotes Multimodal Cyto- and Epigenetic Toxicity.

In this study we evaluated and correlated the cytotoxic effects of zinc oxide nanoparticles (ZnO-NPs) to the epigenetic modifications, using human embryonic kidney (HEK-293) cells as a model system. Imaging of singlet and total reactive oxygen species (ROS) in ZnO-NPs-treated live cells was performed followed by the evaluation of its effects on cytoskeletal, mitochondrial, and nuclear integrity, and on the expression of ROS responsive genes. Next, we determined the global and locus-specific changes in DNA-methylation at the 3 global genomic repeat sequences namely LINE-1, subtelomeric D4Z4 and pericentromeric NBL2, and at the promoter of selected ROS responsive genes (AOX1, HMOX1, NCF2, SOD3). Our studies revealed severe actin depolymerization, increased release of mitochondrial cytochrome C, and nuclear enlargement in ZnO-NPs-treated cells. At the epigenetic level, we observed global reduction in 5-methylcytosine and increase in 5-hydroxymethylcytosine content. Additionally, we observed significant increase in the expression of Ten-Eleven Translocation (TET)-methylcytosine dioxygenase genes but not in the expression of DNA-methyltransferases (DNMTs). Based on our findings, we suggest that ZnO-NPs induce abundant increase in ROS to promote multimodal structural and functional anomalies in cells. Most importantly, ZnO-NP-induced ROS may promote global hypomethylation in cells by triggering the expression of TET-enzymes, avoiding DNMT interferences. Global DNA demethylation is considered to be the hallmark of the majority of cancers and once acquired this could be propagated to future progenies. The present study, hence, can be used as a platform for the assessment of epigenomic toxicity of ZnO-NPs in humans in the light of its use in commercial products.

Tumor Microenvironment-Sensitive Liposomes Penetrate Tumor Tissue via Attenuated Interaction of the Extracellular Matrix and Tumor Cells and Accompanying Actin Depolymerization.

Delivery of anticancer drugs into tumor cores comprised of malignant cancer cells can result in potent therapeutic effects. However, conventional nanoparticle-based drug delivery systems used for cancer therapy often exhibit inefficient tumor-penetrating properties, thus, suggesting the need to improve the functional design of such systems. Herein, we focus on the interactions between cancer cells and the extracellular matrix (ECM) and demonstrate that liposomes modified with slightly acidic pH-sensitive peptide (SAPSp-lipo) can penetrate in vivo tumor tissue and in vitro spheroids comprised of cancer cells and ECM. We previously reported SAPSp-lipo, tumor microenvironment-sensitive liposomes, are effectively delivered to tumor tissue (Hama et al. J Control Release 2015, 206, 67-74). Compared with conventional liposomes, SAPSp-lipo could be delivered to deeper regions within both spheroids and tumor tissues. Furthermore, tumor penetration was found to be promoted at regions where actin depolymerization was induced by SAPSp-lipo and inhibited by the polymerization of actin. In addition, SAPSp-lipo attenuated the interaction between cancer cells and ECM, contributing to the penetration of SAPSp-lipo. These results suggest that SAPSp-lipo penetrates tumors via the interspace route and is accompanied by actin depolymerization. Taken together, SAPSp-lipo demonstrates potential as a novel tumor-penetrable drug carrier for induction of therapeutic effects against malignant cells that comprise tumor cores.

Growing actin networks regulated by obstacle size and shape

Growing actin networks provide the driving force for the motility of cells and intracellular pathogens. Based on the molecular-level processes of actin polymerization, branching, capping, and depolymerization, we have developed a modeling framework to simulate the stochastic and cooperative behaviors of growing actin networks in propelling obstacles, with an emphasis on the size and shape effects on work capacity and filament orientation in the growing process. Our results show that the characteristic size of obstacles changes the protrusion power per unit length, without influencing the orientation distribution of actin filaments in growing networks. In contrast, the geometry of obstacles has a profound effect on filament patterning, which influences the orientation of filaments differently when the drag coefficient of environment is small, intermediate, or large. We also discuss the role of various parameters, such as the aspect ratio of obstacles, branching rate, and capping rate, in affecting the protrusion power of network growth.

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension.

AimsRhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect.Methods and resultsThe analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins.ConclusionsOur study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

Plant actin depolymerizing factor: actin microfilament disassembly and more

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein among eukaryotes. The main function of ADF is the severing and depolymerizing filamentous actin (F-actin), thus regulating F-actin organization and dynamics and contributing to growth and development of the organisms. Mammalian genomes contain only a few ADF genes, whereas angiosperm plants have acquired an expanding number of ADFs, resulting in the differentiation of physiological functions. Recent studies have revealed functions of ADFs in plant growth and development, and various abiotic and biotic stress responses. In biotic stress responses, ADFs are involved in both susceptibility and resistance, depending on the pathogens. Furthermore, recent studies have highlighted a new role of ADF in the nucleus, possibly in the regulation of gene expression. In this review, I will summarize the current status of plant ADF research and discuss future research directions.