RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis.

Polarised targeting of diverse mRNAs to cellular protrusions is a hallmark of cell migration. Although a widespread phenomenon, definitive functions for endogenous targeted mRNAs and their relevance to modulation of in vivo tissue dynamics remain elusive. Here, using single-molecule analysis, gene editing and zebrafish live-cell imaging, we report that mRNA polarisation acts as a molecular compass that orients motile cell polarity and spatially directs tissue movement. Clustering of protrusion-derived RNAseq datasets defined a core 192-nt localisation element underpinning precise mRNA targeting to sites of filopodia formation. Such targeting of the small GTPase RAB13 generated tight spatial coupling of mRNA localisation, translation and protein activity, achieving precise subcellular compartmentalisation of RAB13 protein function to create a polarised domain of filopodia extension. Consequently, genomic excision of this localisation element and perturbation of RAB13 mRNA targeting-but not translation-depolarised filopodia dynamics in motile endothelial cells and induced mispatterning of blood vessels in zebrafish. Hence, mRNA polarisation, not expression, is the primary determinant of the site of RAB13 action, preventing ectopic functionality at inappropriate subcellular loci and orienting tissue morphogenesis.

Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer.

BACKGROUND: Bone marrow metastasis (BMM) is underestimated in gastric cancer (GC). GC with BMM frequently complicate critical hematological abnormalities like diffused intravascular coagulation and microangiopathic hemolytic anemia, which constitute a highly aggressive GC (HAGC) subtype. HAGC present a very poor prognosis with peculiar clinical and pathological features when compared with not otherwise specified advanced GC (NAGC). But the molecular mechanisms underlying BMM from GC remain rudimentary. METHODS: The transcriptomic difference between HAGC and NAGC were analyzed. Genes that were specifically upregulated in HAGC were identified, and their effect on cell migration and invasion was studied. The function of ACTN2 gene were confirmed by GC cell lines, bone-metastatic animal model and patients' tissues. Furthermore, the molecular mechanism of ACTN2 derived-BMM was explored by multiple immunofluorescence staining, western blot, chromatin immunoprecipitation, and luciferase reporter assays. RESULTS: We elucidated the key mechanisms of BMM depending on the transcriptomic difference between HAGC and NAGC. Five genes specifically upregulated in HAGC were assessed their effect on cell migration and invasion. The ACTN2 gene encoding protein α-Actinin-2 was detected enhanced the metastatic capability and induced BMM of GC cells in mouse models. Mechanically, α-Actinin-2 was involved in filopodia formation where it promoted the Actin filament cross-linking by replacing α-Actinin-1 to form α-Actinin-2:α-Actinin-4 complexes in GC cells. Moreover, NF-κB subunit RelA and α-Actinin-2 formed heterotrimers in the nuclei of GC cells. As a direct target of RelA:α-Actinin-2 heterotrimers, the ACTN2 gene was a positive auto-regulatory loop for α-Actinin-2 expression. CONCLUSIONS: We demonstrated a link between filopodia, BMM and ACTN2 activation, where a feedforward activation loop between ACTN2 and RelA is established via actin in response to distant metastasis. Given the novel filopodia formation function and the new mechanism of BMM in GC, we propose ACTN2 as a druggable molecular vulnerability that may provide potential therapeutic benefit against BMM of GC.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ena orchestrates remodelling within the actin cytoskeleton to drive robust Drosophila macrophage chemotaxis.

The actin cytoskeleton is the engine that powers the inflammatory chemotaxis of immune cells to sites of tissue damage or infection. Here, we combine genetics with live in vivo imaging to investigate how cytoskeletal rearrangements drive macrophage recruitment to wounds in Drosophila We find that the actin-regulatory protein Ena is a master regulator of lamellipodial dynamics in migrating macrophages, where it remodels the cytoskeleton to form linear filaments that can then be bundled together by the cross-linker Fascin (also known as Singed in flies). In contrast, the formin Dia generates rare, probing filopods for specialised functions that are not required for migration. The role of Ena in lamellipodial bundling is so fundamental that its overexpression increases bundling even in the absence of Fascin by marshalling the remaining cross-linking proteins to compensate. This reorganisation of the lamellipod generates cytoskeletal struts that push against the membrane to drive leading edge advancement and boost cell speed. Thus, Ena-mediated remodelling extracts the most from the cytoskeleton to power robust macrophage chemotaxis during their inflammatory recruitment to wounds.

Filopodia play an important role in the trans-mesothelial migration of ovarian cancer cells.

Ovarian cancer cells shed from primary tumors can spread easily to the peritoneum via the peritoneal fluid. To allow further metastasis, the cancer cells must interact with the mesothelial cell layer, which covers the entire surface of the peritoneal organs. Although the clinical importance of this interaction between cancer and mesothelial cells has been increasingly recognized, the molecular mechanisms utilized by cancer cells to adhere to and migrate through the mesothelial cell layer are poorly understood. To investigate the molecular mechanisms of cancer cell trans-mesothelial migration, we set up an in vitro trans-mesothelial migration assay using primary peritoneal mesothelial cells. Using this method, we found that downregulation of filopodial protein fascin-1 or myosin X expression in ES-2 cells significantly inhibited the rate of trans-mesothelial migration of cancer cells, whereas upregulation of fascin-1 in SK-OV-3 cells enhanced this rate. Furthermore, downregulation of N-cadherin or integrin ß1 inhibited the rate of cancer cell trans-mesothelial migration. Conversely, downregulation of cortactin or TKS5 or treatment with the MMP inhibitor GM6001 or the N-WASP inhibitor wiskostatin did not have any effect on cancer cell trans-mesothelial migration. These results suggest that filopodia, but not lamellipodia or invadopodia, play an important role in the trans-mesothelial migration of ovarian cancer cells.

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini.

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly.

Filopodia are actin-dependent finger-like structures that protrude from the plasma membrane. Actin filament barbed-end-binding proteins localized to filopodial tips are key to filopodial assembly. Two classes of barbed-end-binding proteins are formins and Ena/VASP proteins, and both classes have been localized to filopodial tips in specific cellular contexts. Here, we examine the filopodial roles of the FMNL formins and Ena/VASP proteins in U2OS cells. FMNL3 suppression reduces filopodial assembly by 90%, and FMNL3 is enriched at >95% of filopodial tips. Suppression of VASP or Mena (also known as ENAH) reduces filopodial assembly by >75%. However, VASP and Mena do not display consistent filopodial tip localization, but are enriched in focal adhesions (FAs). Interestingly, >85% of FMNL3-containing filopodia are associated with FAs. Two situations increase Ena/VASP filopodial localization: (1) expression of myosin-X, and (2) actively spreading cells. In spreading cells, filopodia often mark sites of nascent adhesions. Interestingly, VASP suppression in spreading cells causes a significant increase in adhesion assembly at filopodial tips. This work demonstrates that, in U2OS cells, Ena/VASP proteins play roles in filopodia beyond those at filopodial tips.This article has an associated First Person interview with the first author of the paper.

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Long noncoding RNA UCA1 enhances sensitivity to oncolytic vaccinia virus by sponging miR-18a/miR-182 and modulating the Cdc42/filopodia axis in colorectal cancer.

The promising anti-tumor effects of oncolytic vaccinia virus (OVV) have been demonstrated. Further, we previously showed that long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) enhances OVV cell-to-cell spread via the activation of Cdc42 in ovarian cancer. However, its role in other cancer types and the molecular mechanism underlying its effects remain to be explored. In this study, we first demonstrated that UCA1 upregulates OVV cell-to-cell spread but not its binding, entry, and replication in colorectal cancer cells. Functional analysis indicated that Cdc42 activation and filopodia formation play an important role in this process. Moreover, expression analysis of various miRNAs suggested that UCA1 inhibits both miR-18a and miR-182, thereby promoting Cdc42 activation, which in turn, regulates OVV cell-to-cell spread. Furthermore, UCA1 was found to modulate tumor malignancy, drug resistance, and sensitivity to OVV via different miRNAs in colorectal cancer. These findings indicate that a three-marker panel, which includes UCA1 expression, Cdc42 activation, and filopodia formation, could potentially be used to predict the therapeutic effect of OVV in colorectal cancer.

Baicalein disturbs the morphological plasticity and motility of breast adenocarcinoma cells depending on the tumor microenvironment.

During tumor invasion, cancer cells change their morphology and mode of migration based on communication with the surrounding environment. Numerous studies have indicated that paracrine interactions from non-neoplastic cells impact the migratory and invasive properties of cancer cells. Thus, these interactions are potential targets for anticancer therapies. In this study, we showed that the flavones member baicalein suppresses the motility of breast cancer cells that is promoted by paracrine interactions. First, we identified laminin-332 (LN-332) as a principle paracrine factor in conditioned medium from mammary epithelium-derived MCF10A cells that regulates the morphology and motility of breast adenocarcinoma MDA-MB-231 cells. Then, we carried out a morphology-based screen for small compounds, which showed that baicalein suppressed the morphological changes and migratory activity of MDA-MB-231 cells that were induced by conditioned medium from MCF10A cells and LN-332. We also found that baicalein caused narrower and incomplete lamellipodia formation in conditioned medium-treated MDA-MB-231 cells, although actin dynamics downstream of Rho family small GTPases were unaffected. These results suggest the importance of mammary epithelial cells in the cancer microenvironment promoting the migratory activity of breast adenocarcinoma cells and show a novel mechanism through which baicalein inhibits cancer cell motility.

The 17,18-epoxyeicosatetraenoic acid-G protein-coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques.

BACKGROUND: Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE) is a recently identified new class of antiallergic and anti-inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. OBJECTIVE: We evaluated the effectiveness of 17,18-EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18-EpETE. METHODS: Contact hypersensitivity was induced by topical application of 2,4-dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. Neutrophil mobility was examined by means of imaging analysis in vivo and neutrophil culture in vitro. The receptor for 17,18-EpETE was identified by using the TGF-α shedding assay, and the receptor's involvement in the anti-inflammatory effects of 17,18-EpETE was examined by using KO mice and specific inhibitor treatment. RESULTS: We found that preventive or therapeutic treatment with 17,18-EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18-EpETE was recognized by G protein-coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant-induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18-EpETE was abolished in the absence or inhibition of GPR40. CONCLUSION: 17,18-EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases.

Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fiber formation without inducing epithelial-to-mesenchymal transition.

Aquaporin-5 (AQP5) is a plasma membrane water channel mainly expressed in secretory glands. Increased expression of AQP5 is observed in multiple cancers, including breast cancer, where high expression correlates with the degree of metastasis and poor prognosis. Moreover, studies in cancer cells have suggested that AQP5 activates Ras signaling, drives morphological changes, and in particular increased invasiveness. To design intervention strategies, it is of utmost importance to characterize and dissect the cell biological changes induced by altered AQP5 expression. To isolate the effect of AQP5 overexpression from the cancer background, AQP5 was overexpressed in normal epithelial MDCK cells which have no endogenous AQP5 expression. AQP5 overexpression promoted actin stress fiber formation and lamellipodia dynamics. Moreover, AQP5 decreased cell circularity. Phosphorylation of AQP5 on serine 156 in the second intracellular loop has been shown to activate the Ras pathway. When serine 156 was mutated to alanine to mimic the nonphosphorylated state, the decrease in cell circularity was reversed, indicating that the AQP5-Ras axis is involved in the effect on cell shape. Interestingly, the cellular changes mediated by AQP5 were not associated with induction of epithelial-to-mesenchymal transition. Thus, AQP5 may contribute to cancer by altering cellular morphology and actin organization, which increase the metastatic potential.

Acidic pHe regulates cytoskeletal dynamics through conformational integrin ß1 activation and promotes membrane protrusion.

An acidic extracellular pH (pHe) in the tumor microenvironment has been suggested to facilitate tumor growth and metastasis. However, the molecular mechanisms by which tumor cells sense acidic signal to induce a transition to an aggressive phenotype remain elusive. Here, we showed that an acidic pHe (pH 6.5) stimulation resulted in protrusion and epithelial-mesenchymal transition (EMT) of cancer cells, which promoted migration and matrix degeneration. Using computational molecular dynamics simulations, we reported acidic pHe-induced opening of the Integrin dimers (α5ß1) headpiece which indicated the activation of integrin. Moreover, acidic pHe promoted maturation of focal adhesions, temporal activation of Rho GTPases and microfilament reorganization through integrin ß1-activated FAK signaling. Furthermore, mechanical balance of cytoskeleton (actin, tubulin and vimentin) contributed to acidic pHe-triggered protrusion and morphology change. Taken together, these findings revealed that integrin ß1 could be a novel pH-regulated sensitive molecule which confers protrusion and malignant phenotype of cancer cells.

Sphingolipids Signaling in Lamellipodia Formation and Enhancement of Endothelial Barrier Function.

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury.

Accumulation of glucosylceramide in the absence of the beta-glucosidase GBA2 alters cytoskeletal dynamics.

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.

Submandibular gland pleomorphic adenoma: Histopathological capsular characteristics and correlation with the surgical outcome.

Pleomorphic adenoma (PA) of the submandibular gland is known to have a very low recurrence rate. The aim of this study was to investigate the histopathological and capsular characteristics of submandibular gland PA, looking for any differences between submandibular PA and the reported data for PA of the parotid gland as possible explanation for its low recurrence rate. We reviewed 72 submandibular gland PAs resected at our center between 2000 and 2016. Patient age ranged from 14 to 77 years (mean, 47.2). At least follow (range, 12 to 170 months; mean, 82), none of the 72 patients developed a local recurrence. Histologically, all of the tumors were encased by a complete and intact anatomical capsule (100%). Pseudopodia were detected in 11 (15.3%) and satellite nodules in 3 (4.2%) cases. The histological subtype (according to Seifert et al.) was classic (mixed) in 39 (54.2%), stroma-rich/myxoid in 18/72 (25%) and cellular in 15 (20.8%) cases. A complete rim of healthy pericapsular tissue encasing the tumor and its capsule was observed in only 23/72 (31.9%) cases. In conclusion, submandibular PAs are characterized by consistent presence of an intact anatomical capsule, infrequent occurrence of pseudopodia, a remarkably infrequent occurrence lower frequency of secondary satellite tumor nodules and a comparatively lower proportion of the fragile risky myxoid subtype. Despite the fact that surgery of the submandibular gland can frequently lead to focal capsular exposure, the aforementioned capsular characteristics of submandibular gland PA are probably responsible for the excellent oncologic results.

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.

Comparison of ultrastructural and nanomechanical signature of platelets from acute myocardial infarction and platelet activation.

Acute myocardial infarction (AMI) initiation and progression follow complex molecular and structural changes in the nanoarchitecture of platelets. However, it remains poorly understood how the transformation from health to AMI alters the ultrastructural and biomechanical properties of platelets within the platelet activation microenvironment. Here, we show using an atomic force microscope (AFM) that platelet samples, including living human platelets from the healthy and AMI patient, activated platelets from collagen-stimulated model, show distinct ultrastructural imaging and stiffness profiles. Correlative morphology obtained on AMI platelets and collagen-activated platelets display distinct pseudopodia structure and nanoclusters on membrane. In contrast to normal platelets, AMI platelets have a stiffer distribution resulting from complicated pathogenesis, with a prominent high-stiffness peak representative of platelet activation using AFM-based force spectroscopy. Similar findings are seen in specific stages of platelet activation in collagen-stimulated model. Further evidence obtained from different force measurement region with activated platelets shows that platelet migration is correlated to the more elasticity of pseudopodia while high stiffness at the center region. Overall, ultrastructural and nanomechanical profiling by AFM provides quantitative indicators in the clinical diagnostics of AMI with mechanobiological significance.

Fascin phosphorylation sites combine to regulate esophageal squamous cancer cell behavior.

Filopodia are dynamic membrane extensions generated by F-actin bundling and are involved in cancer cell migration, invasion and metastasis. Fascin is the crucial actin-bundling protein in filopodia, with phosphorylation at fascin serine 39 being well characterized to regulate fascin-mediated actin bundling in filopodia. However, increasing evidence indicates that fascin is phosphorylated at a number of sites. Whether phosphorylation at other sites also regulates fascin function is unknown. In this study, we show that four potential phosphorylation sites in fascin, specifically tyrosine 23, serine 38, serine 39 and serine 274, regulate cell behavior and filopodia formation in esophageal squamous cancer cells. Expression of non-phosphorylatable mutations at each of the four sites promoted anchorage-independent growth, cell motility and filopodia formation, whereas phosphomimetic mutations at each of these sites inhibited these cell behaviors, implying that fascin function in esophageal squamous cancer is regulated by fascin phosphorylation at multiple sites. Furthermore, phosphorylation at S38 and S39 cooperatively regulated cell behavior and filopodia formation, with dual dephosphorylation at both S38 and S39 residues maximally enhancing cell proliferation, migration and filopodia formation, and phosphorylation at any of the two phosphorylatable sites resulting in reduced enhancement. Taken together, our results reveal that phosphorylation at fascin amino acids Y23, S38, S39 and S274, in combination, downregulates the extent of anchorage-independent growth, cell migration and filopodia formation in esophageal squamous cancer cells.

LMO2 Enhances Lamellipodia/Filopodia Formation in Basal-Type Breast Cancer Cells by Mediating ARP3-Profilin1 Interaction.

BACKGROUND The human LMO2 gene was first cloned from an acute T lymphocytic leukemia patient; it is primarily expressed in hematopoietic and vascular endothelial systems, and functions as a pivotal transcriptional regulator during embryonic hematopoiesis and angiogenesis. However, some recent reports indicated that LMO2 is widely expressed in many tissues and tumors, predominantly in cytoplasm, and revealed complicated functions on tumor behaviors in a variety of cancer types. As an adaptor molecule, binding partners and function details of LMO2 in these solid tumors need to be further investigated. MATERIAL AND METHODS In this study, we used yeast two-hybrid method to screen potential LMO2 interacting partners, MBP-pulldown, and co-immunoprecipitation assay to confirm protein-protein interactions, and confocal microscopy to reveal the subcellular localization of relevant proteins and actin cytoskeleton changes in relevant cells. RESULTS We found that ARP3 and profilin1 were 2 binding partners of LMO2, primarily in cytoplasm. LMO2. Functionally, LMO2 mediated the assembly of a complex including ARP3, profilin1, and actin monomer, increased actin monomer binding to profilin1, and promoted lamellipodia/filopodia formation in basal-type breast cancer cells. CONCLUSIONS Our data indicate a novel functional mechanism of LMO2 in facilitating the delivery of actin monomers to the branched microfilament and increasing lamellipodia/filopodia formation in basal-type breast cancer cells, suggesting a cancer-promoting role of LMO2 in a subtype-dependent manner and its potential as a subtype-specific biomarker for clinical treatment of breast cancers.