Actin-bundling Protein Research Articles

Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus.

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key "switch" in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the "S5A" mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host-pathogen defense interactions.

Downregulation of fascin in the first trimester placental villi is associated with early recurrent miscarriage

Inadequate trophoblast proliferation, shallow invasion and exaggerated rate of trophoblast apoptosis are implicated in early recurrent miscarriage (ERM). However, the mechanistic bases of this association have not been fully established. We aimed at investigating the involvement of fascin, an actin-bundling protein, in trophoblast activities and ERM. We found that fascin was downregulated in the cytotrophoblasts (CTBs) and distal cytotrophoblasts (DCTs) of ERM placentae. Knockdown of fascin altered cellular and nucleolar morphology, and inhibited the proliferation but increased apoptosis of trophoblastic HTR8/SVneo cells. Furthermore, fascin knockdown decreased the expression of transcription factors such as Snail1/2, Twist and Zeb1/2, mesenchymal molecules such as Vimentin and N-cadherin, and the protein expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylates signal transducer and activator of transcript 3 (STAT3). Exposure of HTR-8/SVneo cells to hypoxia reoxygenation (H/R) decreased fascin expression to affect the cells’ invasion. Our results indicate for the first time that the downregulation of fascin is involved in the pathogenesis of early recurrent miscarriage; and hence a potential therapeutic target against the disease.

Recapitulating Actin Module Organization in the Drosophila Oocyte Reveals New Roles for Bristle-Actin-Modulating Proteins.

The generation of F-actin bundles is controlled by the action of actin-binding proteins. In Drosophila bristle development, two major actin-bundling proteins—Forked and Fascin—were identified, but still the molecular mechanism by which these actin-bundling proteins and other proteins generate bristle actin bundles is unknown. In this study, we developed a technique that allows recapitulation of bristle actin module organization using the Drosophila ovary by a combination of confocal microscopy, super-resolution structured illumination microscopy, and correlative light and electron microscope analysis. Since Forked generated a distinct ectopic network of actin bundles in the oocyte, the additive effect of two other actin-associated proteins, namely, Fascin and Javelin (Jv), was studied. We found that co-expression of Fascin and Forked demonstrated that the number of actin filaments within the actin bundles dramatically increased, and in their geometric organization, they resembled bristle-like actin bundles. On the other hand, co-expression of Jv with Forked increased the length and density of the actin bundles. When all three proteins co-expressed, the actin bundles were longer and denser, and contained a high number of actin filaments in the bundle. Thus, our results demonstrate that the Drosophila oocyte could serve as a test tube for actin bundle analysis.

Fascin inhibitor increases intratumoral dendritic cell activation and anti-cancer immunity

SUMMARYFascin protein is the main actin-bundling protein in filopodia and invadopodia, which are critical for tumor cell migration, invasion, and metastasis. Small-molecule fascin inhibitors block tumor invasion and metastasis and increase the overall survival of tumor-bearing mice. Here, we report a finding that fascin blockade additionally reinvigorates anti-tumor immune response in syngeneic mouse models of various cancers. Fascin protein levels are increased in conventional dendritic cells (cDCs) in the tumor microenvironment. Mechanistically, fascin inhibitor NP-G2–044 increases the number of intratumoral-activated cDCs and enhances the antigen uptake by cDCs. Furthermore, together with PD-1 blocking antibody, NP-G2–044 markedly increases the number of activated CD8+ T cells in the otherwise anti-PD-1 refractory tumors. Reduction of fascin levels in cDCs, but not fascin gene knockout in tumor cells, mimics the anti-tumor immune effect of NP-G2–044. These data demonstrate that fascin inhibitor NP-G2–044 simultaneously limits tumor metastasis and reinvigorates anti-tumor immune responses.

Rab35 regulates skeletogenesis and gastrulation by facilitating actin remodeling and vesicular trafficking.

Rab35 is a small GTPase that regulates plasma membrane to early endosome vesicular trafficking and mediates actin remodeling to form actin-rich cellular structures. While the function of Rab35 in the cellular context has been examined, its role during development has not been well studied. In this study, we take advantage of the sea urchin's high fecundity, external fertilization, and transparent embryos to determine the function of Rab35 during development. We found that loss of function of Rab35 results in defects in skeletogenesis and gastrulation, which were rescued by co-injection of sea urchin Rab35. The loss of Rab35's function results in decreased endocytosis and impaired exocytosis, which may be important for skeletogenesis and gastrulation. Skeletal spicules of Rab35 knockdown embryos have reduced organized actin compared to the control, supporting the notion that Rab35 regulates actin dynamics. In addition, the skeletal and gastrulation defects induced by Rab35 knockdown were rescued by co-injection with Fascin, an actin-bundling protein, indicating that proper actin dynamics play a critical role for both skeletogenesis and gastrulation. Overall, results indicate that through its role in mediating vesicular trafficking and actin remodeling, Rab35 is an important regulator of embryonic structure formation in early development.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by invivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

L-plastin Ser5 phosphorylation is modulated by the PI3K/SGK pathway and promotes breast cancer cell invasiveness

BackgroundMetastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L-plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L-plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells.MethodsTo unravel the signal transduction network upstream of L-plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhibition/overexpression studies and in vitro kinase assays. To assess the functional impact of L-plastin expression/Ser5 phosphorylation in breast cancer cells, we either silenced L-plastin in cell lines initially expressing endogenous L-plastin or neoexpressed L-plastin wild type and phosphovariants in cell lines devoid of endogenous L-plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis.ResultsOur modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L-plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA-mediated knockdown of L-plastin in BT-20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L-plastin Ser5Glu variant led to increased migration and invasion of BT-549 and MDA-MB-231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L-plastin Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity and concomitant extracellular matrix degradation.ConclusionAltogether, our results demonstrate that L-plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L-plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation.9rn5EeEuriEpmsCBgdyXncVideo abstract

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo.

Simple SummaryColorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Serrated adenocarcinoma (SAC) has been recently recognized by the WHO as a histological CRC with bad prognosis. Consistent with previous evidence, our group identified Fascin1 as a protein directly related to the invasiveness of tumor cells, overexpressed and positively correlated with worse survival in various carcinomas, including SAC. Therefore, Fascin1 has emerged as an ideal target for cancer treatment. In the present study, virtual screening has been carried out from a library of 9591 compounds, thus identifying the FDA-approved anti-retroviral raltegravir (RAL) as a potential Fascin1 blocker. In vitro and in vivo results show that RAL exhibits Fascin1-binding activity and Fascin1-dependent anti-invasive and anti-metastatic properties against CRC cells both in vitro and in vivo.Background: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. Methods: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. Results: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. Conclusion: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner.

LFA-1 cluster formation in T-cells depends on L-plastin phosphorylation regulated by P90RSK and PP2A.

The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.

LYAR Promotes Colorectal Cancer Progression by Upregulating FSCN1 Expression and Fatty Acid Metabolism.

Colorectal cancer (CRC) is a highly malignant tumor associated with poor prognosis, yet the molecular mechanisms are not fully understood. In this study, we showed that LYAR, a nucleolar protein, is expressed at a higher level in CRC tissue than in adjacent normal tissue and that LYAR expression is closely associated with distant CRC metastasis. LYAR not only significantly promotes the migration and invasion of CRC cells in vitro, but knockdown (KD) of LYAR in CRC cells also inhibits xenograft tumor metastasis in vivo. Microarray analysis of LYAR KD cells combined with a chromatin immunoprecipitation (ChIP) assay, gene reporter assay, and rescue experiment indicated that FSCN1 (encoding fascin actin-bundling protein 1 (Fascin-1)) serves as a novel key regulator of LYAR-promoted migration and invasion of CRC cells. Knockdown of FSCN1 significantly inhibits subcutaneous tumorigenesis of CRC cells and leads to the downregulation of FASN and SCD, genes encoding key enzymes in fatty acid synthesis. In summary, this study reveals a novel mechanism by which LYAR promotes tumor cell migration and invasion by upregulating FSCN1 expression and affecting fatty acid metabolism in CRC.

Targeting actin-bundling protein L-plastin as an anabolic therapy for bone loss.

The actin-bundling protein L-plastin (LPL) mediates the resorption activity of osteoclasts, but its therapeutic potential in pathological bone loss remains unexplored. Here, we report that LPL knockout mice show increased bone mass and cortical thickness with more mononuclear tartrate-resistant acid phosphatase-positive cells, osteoblasts, CD31hiEmcnhi endothelial vessels, and fewer multinuclear osteoclasts in the bone marrow and periosteum. LPL deletion impeded preosteoclasts fusion by inhibiting filopodia formation and increased the number of preosteoclasts, which release platelet-derived growth factor-BB to promote CD31hiEmcnhi vessel growth and bone formation. LPL expression is regulated by the phosphatidylinositol 3-kinase/AKT/specific protein 1 axis in response to receptor activator of nuclear factor-κB ligand. Furthermore, we identified an LPL inhibitor, oroxylin A, that could maintain bone mass in ovariectomy-induced osteoporosis and accelerate bone fracture healing in mice. In conclusion, we showed that LPL regulates osteoclasts fusion, and targeting LPL serves as a novel anabolic therapy for pathological bone loss.

Fascin in Cell Migration: More Than an Actin Bundling Protein.

Simple SummaryCell migration is an essential biological process that regulates both development and diseases, such as cancer metastasis. Therefore, understanding the factors that promote cell migration is crucial. One of the factors known to regulate cell migration is the actin-binding protein, Fascin. Fascin is typically thought to promote cell migration through bundling actin to form migratory structures such as filopodia and invadapodia. However, Fascin has many other functions in the cell that may contribute to cell migration. How these novel functions promote cell migration and are regulated is still not well understood. Here, we review the structure of Fascin, the many functions of Fascin and how they may promote cell migration, how Fascin is regulated, and Fascin’s role in diseases such as cancer metastasis.Fascin, an actin-binding protein, regulates many developmental migrations and contributes to cancer metastasis. Specifically, Fascin promotes cell motility, invasion, and adhesion by forming filopodia and invadopodia through its canonical actin bundling function. In addition to bundling actin, Fascin has non-canonical roles in the cell that are thought to promote cell migration. These non-canonical functions include regulating the activity of other actin-binding proteins, binding to and regulating microtubules, mediating mechanotransduction to the nucleus via interaction with the Linker of the Nucleoskeleton and Cytoskeleton (LINC) Complex, and localizing to the nucleus to regulate nuclear actin, the nucleolus, and chromatin modifications. The many functions of Fascin must be coordinately regulated to control cell migration. While much remains to be learned about such mechanisms, Fascin is regulated by post-translational modifications, prostaglandin signaling, protein–protein interactions, and transcriptional means. Here, we review the structure of Fascin, the various functions of Fascin and how they contribute to cell migration, the mechanisms regulating Fascin, and how Fascin contributes to diseases, specifically cancer metastasis.

MiR-125a-5p regulates megakaryocyte proplatelet formation via the actin-bundling protein L-plastin

AbstractThere is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood–derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.

Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

BackgroundUnderstanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood.ResultsWe provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN, and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model.ConclusionsWe propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.

Actin bundles play a different role in shaping scales compared to bristles in the mosquito Aedes aegypti

Insect epithelial cells contain cellular extensions such as bristles, hairs, and scales. These cellular extensions are homologous structures that differ in morphology and function. They contain actin bundles that dictate their cellular morphology. While the organization, function, and identity of the major actin-bundling proteins in bristles and hairs are known, this information on scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito, Aedes aegypti. We show that scales undergo drastic morphological changes during development, from a cylindrical to flat shape with longer membrane invagination. Scale actin-bundle distribution changes from the symmetrical organization of actin bundles located throughout the bristle membrane to an asymmetrical organization. By chemically inhibiting actin polymerization and by knocking out the forked gene in the mosquito (Ae-Forked; a known actin-bundling protein) by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair, and scale morphology. We demonstrated that actin bundles and Ae-Forked are required for bristle elongation, but not for that of scales. In scales, actin bundles are required for width formation. In summary, our results reveal, for the first time, the developmental process of mosquito scale formation and also the role of actin bundles and actin-bundle proteins in scale morphogenesis. Moreover, our results reveal that although scale and bristle are thought to be homologous structures, actin bundles have a differential requirement in shaping mosquito scales compared to bristles.

The Actin Bundling Protein Fascin-1 as an ACE2-Accessory Protein.

We have previously shown that angiotensin-converting enzyme 2 (ACE2), an enzyme counterbalancing the deleterious effects of angiotensin type 1 receptor activation by production of vasodilatory peptides Angiotensin (Ang)-(1–9) and Ang-(1–7), is internalized and degraded in lysosomes following chronic Ang-II treatment. However, the molecular mechanisms involved in this effect remain unknown. In an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ACE2-transfected HEK293T cells. A single protein, fascin-1, was found to differentially interact with ACE2 after Ang-II treatment for 4 h. The interactions between fascin-1 and ACE2 were confirmed by confocal microscopy and co-immunoprecipitation. Overexpression of fascin-1 attenuates the effects of Ang-II on ACE2 activity. In contrast, downregulation of fascin-1 severely decreased ACE2 enzymatic activity. Interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin-1, suggesting that the levels of this protein may change in cardiovascular diseases. In conclusion, we identified fascin-1 as an ACE2-accessory protein, interacting with the enzyme in an Ang-II dependent manner and contributing to the regulation of enzyme activity.

Anti-Metastasis Fascin Inhibitors Decrease the Growth of Specific Subtypes of Cancers.

Fascin is an actin-bundling protein that is critical for filopodial formation and other cellular cytoskeletal structures. An elevated expression of fascin has been observed in tumor cells and is correlated with a shorter survival of cancer patients. Given its roles in tumor cell migration and invasion, we have developed small-molecule fascin inhibitors to prevent and delay tumor metastasis. Here we report the characterization of a new fascin inhibitor in mice. In addition to its inhibitory effects on tumor metastasis, we also report that fascin inhibitors can decrease the growth of specific subtypes of cancers, including epidermal growth factor receptor (EGFR)-high triple-negative breast cancer, and activated B-cell subtypes of diffuse large B-cell lymphoma. Hence, fascin inhibitors can be used to not only inhibit tumor metastasis, but also decrease the tumor growth of specific cancer types.

Identification of the fatty acid synthase interaction network via iTRAQ-based proteomics indicates the potential molecular mechanisms of liver cancer metastasis

BackgroundFatty acid synthase (FASN) is highly expressed in various types of cancer and has an important role in carcinogenesis and metastasis. To clarify the mechanisms of FASN in liver cancer invasion and metastasis, the FASN protein interaction network in liver cancer was identified by targeted proteomic analysis.MethodsWound healing and Transwell assays was performed to observe the effect of FASN during migration and invasion in liver cancer. Isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry were used to identify proteins interacting with FASN in HepG2 cells. Differential expressed proteins were validated by co-immunoprecipitation, western blot analyses and confocal microscopy. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to demonstrate the mechanism of FASN regulating metastasis.ResultsFASN knockdown inhibited migration and invasion of HepG2 and SMMC7721 cells. A total of, 79 proteins interacting with FASN were identified. Additionally, gene ontology term enrichment analysis indicated that the majority of biological regulation and cellular processes that the FASN-interacting proteins were associated with. Co-precipitation and co-localization of FASN with fascin actin-bundling protein 1 (FSCN1), signal-induced proliferation-associated 1 (SIPA1), spectrin β, non-erythrocytic 1 (SPTBN1) and CD59 were evaluated. Knockdown of FASN in liver cancer reduced the expression of FSCN1, SIPA1, SPTBN1 and CD59. Furthermore, inhibition of FASN, FSCN1 or SPTBN1 expression in liver cancer resulted in alterations of epithelial–mesenchymal transition (EMT)-associated markers E-cadherin, N-cadherin, vimentin and transcription factors, Snail and Twist, at the mRNA level, and changes in matrix metallopeptidase (MMP)-2 and MMP-9 protein expression.ConclusionThe results suggested that the FASN-interacting protein network produced by iTRAQ-based proteomic analyses may be involved in regulating invasion and metastasis in liver cancer by influencing EMT and the function of MMPs.

GLABRA2 Regulates Actin Bundling Protein VILLIN1 in Root Hair Growth in Response to Osmotic Stress.

Actin binding proteins and transcription factors are essential in regulating plant root hair growth in response to various environmental stresses; however, the interaction between these two factors in regulating root hair growth remains poorly understood. Apical and subapical thick actin bundles are necessary for terminating rapid elongation of root hair cells. Here, we show that Arabidopsis (Arabidopsis thaliana) actin-bundling protein Villin1 (VLN1) decorates filaments in shank, subapical, and apical hairs. vln1 mutants displayed significantly longer hairs with longer hair growing time and defects in the thick actin bundles and bundling activities in the subapical and apical regions, whereas seedlings overexpressing VLN1 showed different results. Genetic analysis showed that the transcription factor GLABRA2 (Gl2) played a regulatory role similar to that of VLN1 in hair growth and actin dynamics. Moreover, further analyses demonstrated that VLN1 overexpression suppresses the gl2 mutant phenotypes regarding hair growth and actin dynamics; GL2 directly recognizes the promoter of VLN1 and positively regulates VLN1 expression in root hairs; and the GL2-mediated VLN1 pathway is involved in the root hair growth response to osmotic stress. Our results demonstrate that the GL2-mediated VLN1 pathway plays an important role in the root hair growth response to osmotic stress, and they describe a transcriptional mechanism that regulates actin dynamics and thereby modulates cell tip growth in response to environmental signals.

Plastin 1 drives metastasis of colorectal cancer through the IQGAP1/Rac1/ERK pathway.

Tumor metastasis is the dominant cause of death in colorectal cancer (CRC) patients, and it often involves dysregulation of various cytoskeletal proteins. Plastin 1 (PLS1) is an actin‐bundling protein that has been implicated in the structure of intestinal epithelial microvilli; however, its role in CRC metastasis has not yet been determined. In this study, we demonstrated that PLS1 is highly expressed in 33.3% (45/135) of CRC patients and is correlated with lymph node metastasis and poor survival. In in vitro and in vivo experiments, PLS1 induced the migration and invasion of CRC cells and the metastases to the liver and lung in mice. Moreover, the expressions of key factors for CRC metastases, matrix metalloproteinase (MMP) 9 and 2, were enhanced by PLS1, which was dependent on phosphorylating ERK1/2 activated by IQGAP1/Rac1 signaling. The connection between these signals and PLS1 was further confirmed in CRC tissues of patients and the metastatic nodules from a mouse model. These findings suggest that PLS1 promotes CRC metastasis through the IQGAP1/Rac1/ERK pathway. Targeting PLS1 may provide a potential approach to inhibit the metastasis of CRC cells.