Is Your Kid Actin Out? A Series of Six Patients With Inherited Actin-Related Protein 2/3 Complex Subunit 1B Deficiency and Review of the Literature.

BACKGROUND: Hereditary actin-related protein 2/3 complex subunit 1B deficiency is characterized clinically by ear, skin, and lung infections, bleeding, eczema, food allergy, asthma, skin vasculitis, colitis, arthritis, short stature, and lymphadenopathy. OBJECTIVE: We aimed to describe the clinical, laboratory, and genetic features of six patients from four Mexican families. METHODS: We performed exome sequencing in patients of four families with suspected actinopathy, collected their data from medical records, and reviewed the literature for reports of other patients with actin-related protein 2/3 complex subunit 1B deficiency. RESULTS: Six patients from four families were included. All had recurrent infections, mainly bacterial pneumonia, and cellulitis. A total of 67% had eczema whereas 50% had food allergies, failure to thrive, hepatomegaly, and bleeding. Eosinophilia was found in all; 84% had thrombocytopenia, 67% had abnormal-size platelets and anemia. Serum levels of IgG, IgA, and IgE were highly increased in most; IgM was normal or low. T cells were decreased in 67% of patients, whereas B and NK cells were increased in half of patients. Two of the four probands had compound heterozygous variants. One patient was successfully transplanted. We identified 28 other patients whose most prevalent features were eczema, recurrent infections, failure to thrive, bleeding, diarrhea, allergies, vasculitis, eosinophilia, platelet abnormalities, high IgE/IgA, low T cells, and high B cells. CONCLUSION: Actin-related protein 2/3 complex subunit 1B deficiency has a variable and heterogeneous clinical spectrum, expanded by these cases to include keloid scars and Epstein-Barr virus chronic hepatitis. A novel deletion in exon 8 was shared by three unrelated families and might be the result of a founder effect.

Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion.

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

Proline-rich 11 (PRR11) drives F-actin assembly by recruiting the actin-related protein 2/3 complex in human non-small cell lung carcinoma.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100-184 or 100-200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Partial loss of actin nucleator actin-related protein 2/3 activity triggers blebbing in primary T lymphocytes.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.

LncRNA AC009022.1 enhances colorectal cancer cells proliferation, migration, and invasion by promoting ACTR3B expression via suppressing miR-497-5p.

PURPOSE: Long noncoding RNAs (lncRNAs) play an indispensable role in cancer progression. We aim at exploring the effect of AC009022.1 on colorectal cancer (CRC) development in this paper. METHODS: CRC tissues and matched normal tissues of 68 CRC patients were collected. HCT-116 and SW620 cells were transfected and grouped. The cell counting kit-8 assay, cell scratch test, and Transwell experiment were performed to sequentially detect cells proliferation, migration, and invasion. In vivo experiments were conducted. The Luciferase reporter gene assay was used. Gene expression was detected by quantitative reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: AC009022.1 expression was abnormally elevated in CRC tissues and cells. High AC009022.1 expression in CRC patients was significantly associated with poor prognosis. Compared with the siNC group, HCT-116 and SW620 cells of siAC009022.1 group exhibited much lower OD450 value (P < .001) and invasive cell number (P < .01), and significantly higher relative wound width (P < .01). Much lower subcutaneous tumor volume and weight were found in the siAC009022.1 group compared with siNC group (P < .001). In CRC cells, AC009022.1 directly inhibited miR-497-5p expression while miR-497-5p directly hindered ACTR3B expression. Compared with HCT-116 and SW620 cells of oe-AC009022.1 group, those of oe-AC009022.1 + miR-497-5p-mimics group and oe-AC009022.1 + siACTR3B group had obviously lower OD450 value (P < .001) and invasion cell number (P < .01), and markedly higher relative wound width (P < .01). CONCLUSIONS: AC009022.1 enhanced CRC cell proliferation, migration, and invasion by promoting ACTR3B expression via suppressing miR-497-5p.

Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.

Actin related protein 3 (ARP3) promotes apoptosis of intestinal epithelial cells in ulcerative colitis.

Apoptosis in intestinal epithelial cells (IECs) promotes the development of ulcerative colitis (UC), a type of inflammatory bowel disease (IBD). Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Actin related protein 3 (ARP3) promotes endothelial dysfunction. The expression and function of ARP3 in UC remains unclear. In this study, the expression of apoptotic markers as p53, Bax, Cleaved-Caspease9 and Cleaved-Caspease3 were proved to be increased in the intestinal epithelial cells (IECs) of UC patients and in a mouse disuccinimidyl suberate(DSS)-induced colitis model; meanwhile, ARP3 expression was elevated. ARP3 expression levels and the severity of symptoms in patients with UC were positively correlated. By knocking down ARP3 in a TNF-α-treated NCM-460 cell colitis model, the apoptotic markers described above were all decreased. In conclusion, our data indicates that ARP3 might promote the apoptosis of IECs in UC, revealing a potential molecular target for treating UC.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Clinicopathological and prognostic significance of aberrant Arpin expression in gastric cancer.

AIM: To detect the expression of Arpin, and determine its correlation with clinicopathological characteristics and the prognosis of gastric cancer (GC) patients. METHODS: A total of 176 GC patients were enrolled as study subjects and classified into groups according to different clinicopathological variables. GC mucosal tissues were obtained via surgery. Another 43 paraffin-embedded tissue blocks of normal gastric epithelium (> 5 cm away from the edge of the tumor) were included in the control group. Immunohistochemistry (IHC) for the Arpin and Arp3 proteins was performed on the formalin-fixed, paraffin-embedded GC tissues. Additionally, expression of the Arpin protein in 43 normal gastric tissues was also determined using IHC. RESULTS: Expression of the Arpin protein in GC was lower than that in normal gastric mucosa (30.68% vs 60.47%, P < 0.001). A χ2 test of the 176 GC samples used for IHC showed that decreased Arpin expression was associated with advanced TNM stage (P < 0.01) and the presence or absence of lymph node metastasis (80.92% vs 35.56%, P < 0.001). Additionally, a significant correlation was observed between the expression of Arpin and the presence of the Arp2/3 complex in GC tissues (χ2 = 30.535, P < 0.001). Moreover, a multivariate Cox regression analysis revealed that Arpin expression [hazard ratio (HR) = 0.551, P = 0.029] and TNM stage (HR = 5.344, P = 0.001) were independent prognostic markers for overall survival of GC patients. Regarding the 3-year disease-free survival (DFS), the recurrence rate of GC patients with low Arpin expression levels (median DFS 19 mo) was higher than that in the high-Arpin-expression group (median DFS 34 mo, P = 0.022). CONCLUSION: Low Arpin levels are associated with clinicopathological variables and a poor prognosis in GC patients. Arpin may be regarded as a potential prognostic indicator in GC.

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.

The Arp2/3 inhibitory protein Arpin is dispensable for chemotaxis.

Arpin is an Arp2/3 inhibitory protein, which decreases the protrusion lifetime and hence directional persistence in the migration of diverse cells. Arpin is activated by the small GTPase Rac, which controls cell protrusion, thus closing a negative feedback loop that renders the protrusion intrinsically unstable. Because of these properties, it was proposed that Arpin might play a role in directed migration, where directional persistence has to be fine-tuned. We report here, however, that Arpin-depleted tumour cells and Arpin knock-out Dictyostelium amoeba display no obvious defect in chemotaxis. These results do not rule out a potential role of Arpin in other systems, but argue against a general role of Arpin in chemotaxis.

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins.

The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the lamina's major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/ß-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES.

Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex.

Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved.

Planar Cell Polarity (PCP) Protein Vangl2 Regulates Ectoplasmic Specialization Dynamics via Its Effects on Actin Microfilaments in the Testes of Male Rats.

Planar cell polarity (PCP) proteins confer polarization of a field of cells (eg, elongating/elongated spermatids) within the plane of an epithelium such as the seminiferous epithelium of the tubule during spermatogenesis. In adult rat testes, Sertoli and germ cells were found to express PCP core proteins (eg, Van Gogh-like 2 [Vangl2]), effectors, ligands, and signaling proteins. Vangl2 expressed predominantly by Sertoli cells was localized at the testis-specific, actin-rich ectoplasmic specialization (ES) at the Sertoli-spermatid interface in the adluminal compartment and also Sertoli-Sertoli interface at the blood-testis barrier (BTB) and structurally interacted with actin, N-cadherin, and another PCP/polarity protein Scribble. Vangl2 knockdown (KD) by RNA interference in Sertoli cells cultured in vitro with an established tight junction-permeability barrier led to BTB tightening, whereas its overexpression using a full-length cDNA construct perturbed the barrier function. These changes were mediated through an alteration on the organization actin microfilaments at the ES in Sertoli cells, involving actin-regulatory proteins, epidermal growth factor receptor pathway substrate 8, actin-related protein 3, and Scribble, which in turn affected the function of adhesion protein complexes at the ES during the epithelial cycle of spermatogenesis. Using Polyplus in vivo-jetPEI reagent as a transfection medium to silence Vangl2 in the testis in vivo by RNA interference with high efficacy, Vangl2 KD led to changes in F-actin organization at the ES in the epithelium, impeding spermatid and phagosome transport and spermatid polarity, meiosis, and BTB dynamics. For instance, step 19 spermatids remained embedded in the epithelium alongside with step 9 and 10 spermatids in stages IX-X tubules. In summary, the PCP protein Vangl2 is an ES regulator through its effects on actin microfilaments in the testis.

Gelsolin interacts with LamR, hnRNP U, nestin, Arp3 and ß-tubulin in human melanoma cells as revealed by immunoprecipitation and mass spectrometry.

Gelsolin, a multifunctional actin binding protein, plays a not yet fully understood role in tumorigenesis. Therefore the goal of this study was to identify additional molecular partners of gelsolin in human melanoma cells, separately in the cytoplasmic compartment and cell nuclei. For this purpose we performed immunoprecipitation experiments based on a modified protocol followed by mass spectrometry. The obtained results were confirmed by Western blot analysis, proximity ligation assays and confocal microscopy. As expected gelsolin interacted with actin, in particular we demonstrate its interaction with cytoplasmic ß and γ actins, and a newly discovered actin isoform, actbl2. As new gelsolin-interacting partners we identified the ribosomal protein Rpsa, also known as a non-integrin laminin receptor (LamR), and the heterogeneous nuclear ribonucleoprotein hnRNP U. Our data furthermore indicate that gelsolin interacts with particular components of the three cytoskeleton systems: nestin (intermediate filaments), Arp3 (actin cytoskeleton) and ß-tubulin (microtubules). We also report for the first time that gelsolin is a constituent of midbodies, a tubulin containing structure formed at the end of cytokinesis.

α5ß1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3.

Invasive migration in 3D extracellular matrix (ECM) is crucial to cancer metastasis, yet little is known of the molecular mechanisms that drive reorganization of the cytoskeleton as cancer cells disseminate in vivo. 2D Rac-driven lamellipodial migration is well understood, but how these features apply to 3D migration is not clear. We find that lamellipodia-like protrusions and retrograde actin flow are indeed observed in cells moving in 3D ECM. However, Rab-coupling protein (RCP)-driven endocytic recycling of α5ß1 integrin enhances invasive migration of cancer cells into fibronectin-rich 3D ECM, driven by RhoA and filopodial spike-based protrusions, not lamellipodia. Furthermore, we show that actin spike protrusions are Arp2/3-independent. Dynamic actin spike assembly in cells invading in vitro and in vivo is regulated by Formin homology-2 domain containing 3 (FHOD3), which is activated by RhoA/ROCK, establishing a novel mechanism through which the RCP-α5ß1 pathway reprograms the actin cytoskeleton to promote invasive migration and local invasion in vivo.

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer.

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration.

Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge.

RasGRP3 regulates the migration of glioma cells via interaction with Arp3.

Glioblastoma (GBM), the most aggressive primary brain tumors, are highly infiltrative. Although GBM express high Ras activity and Ras proteins have been implicated in gliomagenesis, Ras-activating mutations are not frequent in these tumors. RasGRP3, an important signaling protein responsive to diacylglycerol (DAG), increases Ras activation. Here, we examined the expression and functions of RasGRP3 in GBM and glioma cells. RasGRP3 expression was upregulated in GBM specimens and glioma stem cells compared with normal brains and neural stem cells, respectively. RasGRP3 activated Ras and Rap1 in glioma cells and increased cell migration and invasion partially via Ras activation. Using pull-down assay and mass spectroscopy we identified the actin-related protein, Arp3, as a novel interacting protein of RasGRP3. The interaction of RasGRP3 and Arp3 was validated by immunofluorescence staining and co-immunoprecipitation, and PMA, which activates RasGRP3 and induces its translocation to the peri-nuclear region, increased the association of Arp3 and RasGRP3. Arp3 was upregulated in GBM, regulated cell spreading and migration and its silencing partially decreased these effects of RasGRP3 in glioma cells. In summary, RasGRP3 acts as an important integrating signaling protein of the DAG and Ras signaling pathways and actin polymerization and represents an important therapeutic target in GBM.