Assembly Of Actin Cytoskeleton Research Articles

Dragon's blood attenuates LPS-induced intestinal epithelial barrier dysfunction via upregulation of FAK-DOCK180-Rac1-WAVE2-Arp3 and downregulation of TLR4/NF-κB signaling pathways.

Inflammation-induced intestinal epithelial barrier (IEB) dysfunction is one of the important reasons for the occurrence and development of intestinal inflammatory-related diseases, including ulcerative colitis (UC), Crohn's disease and necrotizing enterocolitis (NEC). Dragon's blood (DB) is a traditional Chinese medicine and has been clinically used to treat UC. However, the protective mechanism of DB on intestinal inflammatory-related diseases has still not been elucidated. The present study aimed to explore the protection mechanism of DB on IEB dysfunction in rat ileum and human colorectal adenocarcinoma cells (Caco-2)/human umbilical vein endothelial cells (HUVECs) coculture system induced by lipopolysaccharide (LPS). DB could ameliorate rat ileum mucosa morphological injury, reduce the accumulation of lipid-peroxidation products and increase the expression of junction proteins. DB also alleviated LPS-induced Caco-2 cells barrier integrity destruction in Caco-2/ HUVECs coculture system, leading to increased trans-endothelial electrical resistance (TEER), reduced cell permeability, and upregulation of expressions of F-actin and junction proteins. DB contributed to the assembly of actin cytoskeleton by upregulating the FAK-DOCK180-Rac1-WAVE2-Arp3 pathway and contributed to the formation of intercellular junctions by downregulating TLR4-MyD88-NF-κB pathway, thus reversing LPS-induced IEB dysfunction. These novel findings illustrated the potential protective mechanism of DB on intestinal inflammatory-related diseases and might be useful for further clinical application of DB.

Reverse Mechanotransduction: Driving Chromatin Compaction to Decompaction Increases Cell Adhesion Strength and Contractility.

Mechanical extracellular signals elicit chromatin remodeling via the mechanotransduction pathway, thus determining cellular function. However, the reverse pathway is an open question: does chromatin remodeling shape cells, regulating their adhesion strength? With fluidic force microscopy, we can directly measure the adhesion strength of epithelial cells by driving chromatin compaction to decompaction with chromatin remodelers. We observe that chromatin compaction, induced by performing histone acetyltransferase inhibition or ATP depletion, leads to a reduction in nuclear volume, disrupting actin cytoskeleton and focal adhesion assembly, and ultimately decreases in cell adhesion strength and traction force. Conversely, when chromatin decompaction is drived by removing the remodelers, cells recover their original shape, adhesion strength, and traction force. During chromatin decompaction, cells use depolymerized proteins to restore focal adhesion assemblies rather than neo-synthesized cytoskeletal proteins. We conclude that chromatin remodeling shapes cells, regulating adhesion strength through a reverse mechanotransduction pathway from the nucleus to the cell surface involving RhoA activation.

Cu2−x Se nanoparticles suppress cell proliferation and migration in hepatocellular carcinoma by impairing mitochondrial respiration

Cu2−x Se nanoparticles (Cu2−x Se NPs) as a new therapeutic drug platform is widely used in disease treatment due to their strong near-infrared optical absorption. In recent years, with their continuous expansion of applications in different fields, their own biological effects have received increasing attention. However, little is known about the effect of Cu2−x Se NPs on cancer cell. In this research, we found that Cu2−x Se NPs inhibited proliferation of HepG2 cells (IC50: 15.91 μM) and SMMC-7721 cells (IC50: 43.15 μM) and they mainly induced cell cycle arrest at the G2/M phase. Moreover, Cu2−x Se NPs inhibited HepG2 and SMMC-7721 cell migration and lamellopodia formation. Further studies indicated that Cu2−x Se NPs impaired mitochondrial respiration by inhibiting electron transport chain complex activity, thus reducing adenosine triphosphate levels. The insufficient energy supply subsequently impaired actin cytoskeleton assembly, ultimately inhibiting HepG2 and SMMC-7721 cell proliferation and migration. These findings suggest that Cu2−x Se NPs may have potentially antitumor activity, which might provide new insights of NPs into specific cancer treatment.

CLDN6 inhibits breast cancer metastasis through WIP-dependent actin cytoskeleton-mediated autophagy

BackgroundAs a breast cancer suppressor gene, CLDN6 overexpression was found to inhibit breast cancer metastasis in our previous studies, but the specific mechanism remains unclear. This study aimed to clarify the role and mechanism of CLDN6 in inhibiting breast cancer metastasis.MethodsWestern blot, immunofluorescence and transmission electron microscopy were performed to detect autophagy. Wound healing, transwell assays and lung metastasis mouse models were used to examine breast cancer metastasis. Phalloidin staining and immunofluorescent staining were used to observe actin cytoskeleton. mRNA seq, RT-PCR, western blot, chromatin immunoprecipitation, dual luciferase reporter assay, co-immunoprecipitation and immunofluorescence were performed to define the molecular mechanism. The expression levels and clinical implication of CLDN6, WIP and LC3 in breast cancer tissues were evaluated using immunohistochemistry.ResultsWe demonstrated that CLDN6 inhibited breast cancer metastasis through autophagy in vitro and vivo. We unraveled a novel mechanism that CLDN6 regulated autophagy via WIP-dependent actin cytoskeleton assembly. Through its PDZ-binding motif, overexpressed CLDN6 interacted with JNK and upregulated JNK/c-Jun pathway. C-Jun promoted WIP expression at the transcriptional level. Notably, we observed c-Jun transcriptionally upregulated CLDN6 expression, and there was a positive feedback loop between CLDN6 and JNK/c-Jun. Finally, we found that CLDN6, WIP and LC3 expression correlated with each other, and WIP expression was significantly associated with lymph node metastasis of breast cancer patients.ConclusionsThe data provide a new insight into the inhibitory effects of CLDN6-mediated autophagy on breast cancer metastasis, and revealed the new mechanism of CLDN6 regulating autophagy through WIP-dependent actin cytoskeleton. Our findings enrich the theoretical basis for CLDN6 as a potential biomarker for breast cancer diagnosis and therapy.

Y-27632 Impairs Angiogenesis on Extra-Embryonic Vasculature in Post-Gastrulation Chick Embryos

Y-27632 inhibits Rho-associated coiled-coil-containing protein kinase (ROCK) signaling, which is involved in various embryonic developmental processes, including angiogenesis, by controlling actin cytoskeleton assembly and cell contractility. Administration of Y-27632 impairs cytoskeletal arrangements in post-gastrulation chick embryos, leading to ventral body wall defects (VBWDs). Impaired angiogenesis has been hypothesized to contribute to VBWDs. ROCK is essential in transmitting signals downstream of vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis induces gene expressions and alterations of the actin cytoskeleton upon binding to VEGF receptors (VEGFRs). The aim of this study was to investigate effects of Y-27632 on angiogenesis in post-gastrulation chick embryos during early embryogenesis. After 60 h incubation, embryos in shell-less culture were treated with Y-27632 or vehicle for controls. Y-27632-treated embryos showed reduced extra-embryonic blood vessel formation with impaired circulation of the yolk sac, confirmed by fractal analysis. Western blot confirmed impaired ROCK downstream signaling by decreased expression of phosphorylated myosin light chain. Interestingly, RT-PCR demonstrated increased gene expression of VEGF and VEGFR-2 1 h post-treatment. Protein levels of VEGF were higher in Y-27632-treated embryos at 8 h following treatment, whereas no difference was seen in membranes. We hypothesize that administration of Y-27632 impairs vessel formation during angiogenesis, which may contribute to failure of VWB closure, causing VBWDs.

Elevated barometric pressure suppresses cell proliferation by delaying the G2/M phase and weakening integrin-mediated cell adhesion and actin assembly

Human cells are continuously exposed to various stress factors in their physiological environment. Evidence suggests that certain mechanical stress can affect cell cycle progression and cell proliferation. However, the signaling pathways involved in this process are not well understood. To investigate this, we developed a pressure chamber capable of producing an elevated barometric pressure (EBP) environment of 2?atmospheric absolute pressure (ATA). We then studied the effect of EBP on cell proliferation and its underlying mechanism. Our results show that EBP inhibited cell proliferation by delaying the G2/M phase. Specifically, EBP reduced the expression levels of cell adhesion-related genes and downregulated integrin subunit genes, resulting in weaker interaction between cells and extracellular matrix proteins. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 homolog (Cdc42) activity was suppressed, and actin assembly was decreased. These findings suggest that the EBP-mediated G2/M phase delay is due to attenuated cell adhesion and actin cytoskeleton assembly, leading to the inhibition of cell proliferation. Our results provide a crucial molecular mechanism for how certain pressure (changes) can negatively regulate cell proliferation. These findings could potentially be used in the future to develop a pressure therapy to inhibit cell proliferation in cancer patients.

Effect of mechanical stretching and substrate stiffness on the morphology, cytoskeleton and nuclear shape of corneal endothelial cells

Due to the limited capacity of corneal endothelial cells (CECs) division, corneal endothelial diseases have become a great challenge. The cornea is subjected to various mechanical stimuli in vivo, which may have a positive or negative influence. Thus, it is significant to gain an insight into the mechanism of mechanobiology of CECs for seeking more possible treatment. The purpose of this study was to determine the impacts of mechanical stretch and substrate stiffness on the morphology and fundamental cell behavior of CECs. Rabbit corneal endothelial cells (RCECs) were subjected to a 5% mechanical stretch or cultured on substrates of different stiffness. The impacts of mechanical stimulus on cell area, aspect ratio, circularity, cell density, nuclear shape, cytoskeleton, and cell viability were investigated. The expressions of the corneal endothelium-related markers ZO-1 and Na+/K+-ATPase were also evaluated by confocal immunofluorescence microscopy in the stiffness group. Our results suggested that mechanical stretch promoted the rearrangement of the cytoskeleton while decreasing the cell circularity, nuclear area, and cell density as well as cell viability. RCECs cultured on 10 ​kPa substrates, which was close to the physiological stiffness of rabbit Descemet's membrane (DM), showed better cell morphology, more stable actin cytoskeleton assembly, and more robust expression of the functional marker compared with other softer or stiffer substrates. In summary, mechanical stretch and substrate stiffness have profound influences on the morphology and function of CECs, which may have implications for the understanding and possible treatment of corneal endothelial diseases.

Soft nanofiber modified micropatterned substrates enhance native-like endothelium maturation via CXCR4/calcium-mediated actin cytoskeleton assembly

Soft nanofiber modified micropatterned substrates enhance native-like endothelium maturation via CXCR4/calcium-mediated actin cytoskeleton assembly

Silencing of a Cotton Actin-Binding Protein GhWLIM1C Decreases Resistance against Verticillium dahliae Infection.

LIM proteins are widely spread in various types of plant cells and play diversely crucial cellular roles through actin cytoskeleton assembly and gene expression regulation. Till now, it has not been clear whether LIM proteins function in plant pathogen defense. In this study, we characterized a LIM protein, GhWLIM1C, in upland cotton (Gossypium hirsutum). We found that GhWLIM1C could bind and bundle the actin cytoskeleton, and it contains two LIM domains (LIM1 and LIM2). Both the two domains could bind directly to the actin filaments. Moreover, the LIM2 domain additionally bundles the actin cytoskeleton, indicating that it possesses a different biochemical activity than LIM1. The expression of GhWLIM1C responds to the infection of the cotton fungal pathogen Verticillium dahliae (V. dahliae). Silencing of GhWLIM1C decreased cotton resistance to V. dahliae. These may be associated with the down regulated plant defense response, including the PR genes expression and ROS accumulation in the infected cotton plants. In all, these results provide new evidence that a plant LIM protein functions in plant pathogen resistance and the assembly of the actin cytoskeleton are closely related to the triggering of the plant defense response.

Single-cell atlas of craniogenesis uncovers SOXC-dependent, highly proliferative, and myofibroblast-like osteodermal progenitors.

SUMMARYThe mammalian skull vault is essential to shape the head and protect the brain, but the cellular and molecular events underlying its development remain incompletely understood. Single-cell transcriptomic profiling from early to late mouse embryonic stages provides a detailed atlas of cranial lineages. It distinguishes various populations of progenitors and reveals a high expression of SOXC genes (encoding the SOX4, SOX11, and SOX12 transcription factors) early in development in actively proliferating and myofibroblast-like osteodermal progenitors. SOXC inactivation in these cells causes severe skull and skin underdevelopment due to the limited expansion of cell populations before and upon lineage commitment. SOXC genes enhance the expression of gene signatures conferring dynamic cellular and molecular properties, including actin cytoskeleton assembly, chromatin remodeling, and signaling pathway induction and responsiveness. These findings shed light onto craniogenic mechanisms and SOXC functions and suggest that similar mechanisms could decisively control many developmental, adult, pathological, and regenerative processes.

Abstract 3463: Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to all treatment modalities. Early metastatic dissemination, rich desmoplastic stroma and immunosuppressive infiltrate are some of the features contributing to the poor prognosis of PDAC patients (5-year survival rate of less than 10%). Despite scientific progress in the development of new therapeutic regimes, PDAC patients in low- and middle-income countries have restricted access to high-cost innovative drugs. Recently, attention has been given to the concept of drug repurposing that could meet the need for novel but affordable and widely accessible therapies. Imidazoline receptor 1 Nischarin (NISCH, IR1, IRAS) is an integrin α5-interacting protein that acts as a tumor suppressor in breast cancer through modulation of cancer cell motility and survival. Importantly, NISCH has several clinically approved agonists that are used for the treatment of hypertension and are shown to modulate autophagy and reduce inflammation and fibrosis, which makes NISCH a potentially good therapeutic target for defying PDAC progression. Based on our GSEA analysis of NISCH expression in PDAC patient samples, NISCH was predicted to have an effect on cancer cell focal adhesion assembly and cytoskeletal organization. Therefore, we examined the effects of NISCH agonists on PDAC cells fitness in vitro. A panel of PDAC cell lines was tested in MTT assay to determine the effects of NISCH agonists rilmenidine, clonidine and moxonidine on cell viability. Flow cytometry, western blot and immunofluorescence were used to examine the effects of NISCH agonists on PDAC cell metabolism, migration and cytoskeletal organization. Tg(fli1:EGFP) zebrafish model was used to examine the effects of NISCH agonists on PDAC cell growth and invasion. We found that NISCH was expressed in PDAC cell lines, and that out of the three tested agonists, rilmenidine most potently inhibited cancer cell viability. Treatment with rilmenidine significantly reduced PANC-1, MIA PaCa-2 and BxPC-3 cell attachment to extracellular matrix (collagen type I and fibronectin) and migratory potential in wound healing and transwell assays. Furthermore, treatment with rilmenidine altered the organization of actin cytoskeleton and focal adhesion assembly, potentially through the reduction of intracellular reactive oxygen species (ROS) levels. Importantly, treatment with rilmenidine inhibited invasion of PANC-1 cells in Tg(fli1:EGFP) zebrafish model. With these antimigratory and potentially antimetastatic effects of rilmenidine, our study lays the ground for a more extensive examination of the biological role of NISCH in PDAC progression and implies that its agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Aleksandar Pavic, Tatjana Srdic-Rajic, Jelena Grahovac. Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3463.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10‐phenanthroline, tpphz=tetrapyridophenazine) “RuRuPhen” blocks the transformation of G‐actin monomers to F‐actin filaments with no disassembly of pre‐formed F‐actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G‐actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late‐stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

Analysis of microRNAs in Small Urinary Extracellular Vesicles and Their Potential Roles in Pathogenesis of Renal ANCA-Associated Vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.

PRP4 Induces Epithelial-Mesenchymal Transition and Drug Resistance in Colon Cancer Cells via Activation of p53.

Pre-mRNA processing factor 4B (PRP4) promotes pre-mRNA splicing and signal transduction. Recent studies have shown that PRP4 modulates the assembly of actin cytoskeleton in cancer cells and induces epithelial–mesenchymal transition (EMT) and drug resistance. PRP4 displays kinase domain-like cyclin-dependent kinases and mitogen-activated protein kinases, making it capable of phosphorylating p53 and other target proteins. In the current study, we report that PRP4 induces drug resistance and EMT via direct binding to the p53 protein, inducing its phosphorylation. Moreover, PRP4 overexpression activates the transcription of miR-210 in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner, which activates p53. The involvement of miR-210 in the activation of p53 was confirmed by utilizing si-miR210. si-miR210 blocked the PRP4-activated cell survival pathways and reversed the PRP4-induced EMT phenotype. Moreover, we used deferoxamine as a hypoxia-mimetic agent, and si-HIF to silence HIF-1α. This procedure demonstrated that PRP4-induced EMT and drug resistance emerged in response to consecutive activation of HIF-1α, miR-210, and p53 by PRP4 overexpression. Collectively, our findings suggest that the PRP4 contributes to EMT and drug resistance induction via direct interactions with p53 and actions that promote upregulation of HIF-1α and miR-210. We conclude that PRP4 is an essential factor promoting cancer development and progression. Specific PRP4 inhibition could benefit patients with colon cancer.

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling.

Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes’ CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

Role of Clathrin Light Chains in Regulating Invadopodia Formation.

One of the most fundamental processes of the cell is the uptake of molecules from the surrounding environment. Clathrin-mediated endocytosis (CME) is the best-described uptake pathway and regulates nutrient uptake, protein and lipid turnover at the plasma membrane (PM), cell signaling, cell motility and cell polarity. The main protein in CME is clathrin, which assembles as a triskelion-looking building block made of three clathrin heavy chains and three clathrin light chains. Compared to clathrin heavy chains (CHCs), the role of the two isoforms of clathrin light chains (CLCA and CLCB) is poorly understood. Here, we confirm that the simultaneous deletion of both CLCA/B causes abnormal actin structures at the ventral PM and we describe them, for the first time, as functional invadopodia rather than disorganized actin-cytoskeleton assembly sites. Their identification is based on the occurrence of common invadopodia markers as well as functional invadopodia activity characterized by an increased local proteolytic activity of the extracellular matrix proteins. We demonstrate that CLCA/B deletion impacts the intracellular trafficking and recovery of the matrix metalloproteinase 14 (MMP14) leading to its accumulation at the plasma membrane and induction of invadopodia formation. Importantly, we show that invadopodia formation can be prevented by depletion of MMP14. As such, we propose that CLCA/B regulate invadopodia formation by regulating MMP14 delivery to the plasma membrane.

Effect of Multi-Phosphonate Coating of Titanium Surfaces on Osteogenic Potential.

The aim of this study was to evaluate the impact of a novel multi-phosphonate (MP) coating strategy of dental implant surfaces on the expression of osteogenesis-related factors in vitro. MG-63 human osteoblast-like cells, bone marrow mesenchymal stem cells (BM-MSCs), and human periodontal ligament stem cells (hPDLSCs) were cultured separately on titanium disks with and without MP coating. Cell attachment was visualized by focal adhesion and actin cytoskeleton staining. The proliferation and gene expression of the markers related to osteogenesis and bone turnover were measured after 48 and 120 h of cell culture. Actin cytoskeleton assembly and focal adhesion were similar between test surfaces within each cell type but differed from those on tissue culture plastic (TCP). The proliferation of MG-63 cells and PDLSCs was comparable on all surfaces, while BM-MSCs showed an increase on tissue culture plastic (TCP) versus titanium. The gene expression of osteoprotegerin and receptor activator of nuclear factor-kappa B ligand was higher in MG-63 cells grown on MP-coated surfaces. At the same time, osteocalcin was decreased compared to the other surfaces. Collagen type I gene expression after 120 h was significantly lower in hPDLSCs cultivated on MP-coated surfaces. Within the limitations of this study, MP coating on titanium surfaces might have a slight beneficial effect on bone turnover in vitro.