Focal adhesion dynamics-mediated cell migration and proliferation on silica bead arrays.

E3 ubiqutin ligase for inhibin B receptor (HECTD1) has been demonstrated to play an indispensable role in embryonic development, including neural tube closure, placentation and eye formation, etc. The malformations caused by loss of HECTD1 are also found in humans, such as neural tube closure defects in HECTD1 knockout mouse model are comparable to anencephaly in humans. Therefore, the investigation of HECTD1 function in regulating neural tube closure and placental development may be helpful for understanding the underlying mechanisms of embryonic development and for the search of new approaches to prevent or treat the associated diseases. Normal embryonic development is the result of proper cell movement which is precisely regulated by complex cellular events. Although several mechanisms of HECTD1 function have been proposed recently, many aspects of the connection of HECTD1 with intracellular signaling pathways in cell movement are novel. Using HECTD1 knockout mouse embryonic fibroblasts (MEFs) as the primary model, we first examined cell spreading and migration respectively. We found that loss of HECTD1 shortened the duration and reduced the area of cell spreading, while the velocity was enhanced and the directionality of cell migration impaired. Furthermore, as compared to wild-type cells, the cell adhesion proteins, paxillin and zyxin, were inhibited in maturing from focal complexes into focal adhesions in HECTD1 knockout cells. These defects in the formation of focal adhesions were associated with enhanced tyrosine phosphorylation of paxillin (paxillin-Y118), as well as higher activity of Rac1 and RhoA. Screening with mass spectrometry has led to the identification of IQ motif containing GTPase activating protein 1 (IQGAP1) which is essential for the formation of cell polarity, stabilization of cell-cell adhesion and proper cell movement (Watanabe et al., 2004; Kuroda et al., 1998; Choi et al., 2013). Interestingly, we discovered that, in contrast to wild-type cells, IQGAP1 is overexpressed in HECTD1 knockout cells. Using co-immunoprecipitation and co-localization assay, we confirmed that IQGAP1 and HECTD1 physically interact with each other. Since HECTD1 is an E3 ubiquitin ligase, then we examined whether IQGAP1 acts as the substrate protein of HECTD1 by checking the ubiquitination and half life of IQGAP1. Our results confirmed that in HECTD1 knockout cells ubiquitination was reduced thereby prolonging the half life of IQGAP1. To answer the question whether the changes in adhesion proteins and cell movement observed in HECTD1 knockout cells were caused by overexpression of IQGAP1, we mimicked the observed effects by overexpressing GFP-IQGAP1 in wild-type cells and by siRNA knockdown of IQGAP1 in HECTD1 knockout cells. Intriguingly, overexpression of GFP-IQGAP1 in wild-type cells resulted in similar impaired expression of focal adhesions in HECTD1 knockout cells, while siRNA knockdown of IQGAP1 in HECTD1 knockout cells significantly rescued the maturation of focal adhesions, activity of RhoA, duration of cell spreading and velocity of cell migration. Taken all data together, our findings indicate that HECTD1 plays a regulatory role in ubiquitination of IQGAP1, which in turn impacts on dynamics of focal adhesions and regulates cell spreading and migration.

Directional cell migration drives embryonic development, cancer metastasis, and tissue repair and regeneration. Here, we examine the role of intraflagellar transport (IFT) 20 (Ift20) during polarized migration of epidermal cells. IFT20 is implicated in regulating cell migration independently of the primary cilium, but how IFT proteins integrate with the cell migration machinery is poorly understood. We show that genetic ablation of IFT20 in vitro slows keratinocyte migration during wound healing. We find that this phenotype is independent of the primary cilium and instead can be attributed to alterations in integrin-mediated mechanotransduction and focal adhesion (FA) dynamics. Loss of Ift20 resulted in smaller and less numerous FAs and reduced the levels of activated FA kinase. Studies of FA dynamics during microtubule-induced FA turnover demonstrated that Ift20 loss specifically impaired the reformation, but not the disassembly, of FAs. In the absence of Ift20 function, β1 integrins endocytosed during FA disassembly are not transferred out of Rab5 (+) endosomes. This defective transit from the early endosome disrupts eventual recycling of β1 integrins back to the cell surface, resulting in defective FA reformation. In vivo, conditional ablation of Ift20 in hair follicle stem cells (HF-SCs) similarly impairs their ability to invade and migrate during epidermal wound healing. Using explant studies, lineage tracing, and clonal analysis, we demonstrate that Ift20 is required for HF-SC migration and their contribution to epidermal regeneration. This work identifies a new Ift20 mechanotrafficking mechanism required for polarized cell migration and stem cell–driven tissue repair.

Biomaterial-driven modulation of cell adhesion and migration is a challengingaspect of tissue engineering. Here, we investigated the impact of surface-boundmicrogel arrays with variable geometry and adjustable cross-linking propertieson cell adhesion and migration. We show that cell migration is inverselycorrelated with microgel array spacing, whereas directionality increases asarray spacing increases. Focal adhesion dynamics is also modulated by microgeltopography resulting in less dynamic focal adhesions on surface-bound microgels.Microgels also modulate the motility and adhesion of Sertoli cells used as amodel for cell migration and adhesion. Both focal adhesion dynamics and speedare reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeletonthat mediates the interaction between microtubules and microfilaments, isdispensable for the regulation of cell adhesion and migration on microgels.Finally, increasing microgel cross-linking causes a clear reduction of focaladhesion turnover in Sertoli cells. These findings not only show that spacingand rigidity of surface-grafted microgels arrays can be effectively used tomodulate cell adhesion and motility of diverse cellular systems, but they alsoform the basis for future developments in the fields of medicine and tissueengineering.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

BackgroundThe long noncoding RNA (lncRNA) JPX is a molecular switch for X-chromosome inactivation. Accumulating studies have shown that the aberrant expression and function of lncRNAs are involved in the occurrence and development of tumors. However, the functional importance and mechanism of the action of lncRNA JPX in cervical cancer (CC) remain unknown.MethodIn this study, qRT-PCR and western blotting were used to evaluate the mRNA or protein expression of JPX, miR-25-3p and SOX4 in CC tissues and cell lines. StarBase v2.0 database, luciferase reporter assay and RNA immunoprecipitation assay were used to explore the relationship between JPX and miR-25-3p. EdU assay, CCK-8 assay and transwell assay were utilized to evaluate the proliferation, migration and invasion of CC cells. The tumor xenograft assay in nude mice was performed to demonstrate the role of the JPX/miR-25-3p/SOX4 axis in CC.ResultsWe found that JPX was markedly upregulated, whereas miR-25-3p was markedly downregulated in CC tissues and cell lines, and the expression of JPX was negatively correlated with miR-25-3p in CC tissues. Moreover, overexpression of JPX increased proliferation, migration and invasion of HeLa cells, whereas knockdown of JPX decreased proliferation, migration and invasion of HeLa cells. In contrast to JPX, overexpression of miR-25-3p decreased proliferation, migration and invasion of HeLa cells. In addition, knockdown of JPX was found to inhibit HeLa cell viability and tumor development via up-regulating the expression of miR-25-3p and inhibiting the expression of SOX4.ConclusionsOur study demonstrates that JPX promotes cervical cancer progression through modulating the miR-25-3p/SOX4 axis, and may serve as a potential target for CC therapy.

An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (∼1140 molecules/µm2) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays.

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors.

In recent years, the incidence and mortality of cervical cancer are increasing worldwide. At the same time, more and more data have confirmed that miRNA-mRNA plays a positive or negative regulatory role in many cancers. This study attempted to screen effective miRNA-mRNA in the progression of cervical cancer, and to study the mechanism of miRNA-mRNA in the progression of cervical cancer. The expression profile data of GSE7410, GSE 63514, GSE 86100 and TCGA-CESC were downloaded, and 34 overlapping differentially expressed genes (22 up regulated and 12 down regulated) and 166 miRNAs (74 down regulated and 92 up regulated) were screened through limma package. Then, miR-197-3p/TYMS pairs were obtained by PPI, functional enrichment, Kaplan Meier plotter analysis, Cox univariate and multivariate analysis, risk modeling, WGCNA, qPCR and double luciferase experiments. The results showed that TYMS was an independent prognostic factor of cervical cancer, and its expression level was negatively correlated with cervical cancer tissue grade (TMN), tumor grade, age, microsatellite stability and tumor mutation load, and positively correlated with methyl expression in DNMT1, DNMT2, DNMT3A and DNMT3B. Functional experiments showed that TYMS knockout could promote the proliferation, migration and invasion of HeLa cells and reduce apoptosis. Overexpression of TYMS showed the opposite trend. miR-197-3p was negatively correlated with the expression of TYMS. MiR-197-3p inhibitor could reverse the effect of si-TYMS on the proliferation of HeLa cells. In conclusion, these results reveal that TYMS plays a very important role in the prognosis and progression of cervical cancer, and has the potential to be called cervical cancer biomarkers. At the same time, miR-197-3p/TYMS axis can regulate the deterioration of cervical cancer cells, which lays a foundation for the molecular diagnosis and treatment of cervical cancer.

Focal adhesion (FA) play a critical role in many biological processes which include cell survival and cell migration. They serve as cellular anchor, allowing cells to stay attached to the extracellular matrix (ECM), and can also regulate cellular transduction. Previously, it has been suggested that vesicles such as endosomes could interact directly with FA or be implicated in their turnover. In this study, we investigated whether there is a relationship between FA and the early endocytic machinery in MDA-MB-231 cells. In this study, cell culture, transfection, time laps confocal microscopies, immunocytochemistry, western blotting, Cell fractionation and immunoprecipitation techniques were performed. Cells acutely treated with Dynasore, an inhibitor of dynamin, or with Pitstop 2, an inhibitor of clathryn-dependent endocytosis showed a reduction in the expression of early endosome biomarkers such as Rab5 and EEA1. Additionally, cells treated with these endocytic inhibitors exhibited an increase number and size of FA, as well as an increase FA turnover duration. This data was consistent with the reduction of the speed of cell migration. We demonstrated that Rab5- and EEA1-positive early endosomes were found to be colocalized with internalized FA. The present study suggests that there is a link between FA and early endosome markers, which indicates that the early endosomes may be involved in FA dynamics.

Smad nuclear interacting protein 1 (SNIP1) is a nuclear protein and involved in essential biological processes. MicroRNAs are effective regulators of tumorigenesis and cancer progression via targeting multiple genes. In present study, we aimed to investigate the function of SNIP1 and identify novel miRNA-SNIP1 axis in the development of cervical cancer. The results showed for the first time that silencing of the SNIP1 gene inhibited the migration and proliferation in HeLa cells significantly. Bioinformatics analysis and dual luciferase reporter assay demonstrated that miR-29a-3p could target 3′ UTR of SNIP1 directly. The mRNA and protein expression levels of SNIP1 were negative regulated by miR-29a-3p according to the RT-qPCR and Western blot analysis, respectively. Furthermore, functional studies showed that over-expression of miR-29a-3p restrained HeLa cells migration and proliferation, and the mRNA expression of SNIP1 downstream genes (HSP27, c-Myc, and cyclin D1) were down-regulated by miR-29a-3p. Together, we concluded that miR-29a-3p suppressed the migration and proliferation in HeLa cells by directly targeting SNIP1. The newly identified miR-29a-3p/SNIP1 axis could provide new insight into the development of cervical cancer.

The spectraplakin family of proteins includes ACF7/MACF1 and BPAG1/dystonin in mammals, VAB-10 in Caenorhabditis elegans, Magellan in zebrafish, and Short stop (Shot), the sole Drosophila member. Spectraplakins are giant cytoskeletal proteins that cross-link actin, microtubules, and intermediate filaments, coordinating the activity of the entire cytoskeleton. We examined the role of Shot during cell migration using two systems: the in vitro migration of Drosophila tissue culture cells and in vivo through border cell migration. RNA interference (RNAi) depletion of Shot increases the rate of random cell migration in Drosophila tissue culture cells as well as the rate of wound closure during scratch-wound assays. This increase in cell migration prompted us to analyze focal adhesion dynamics. We found that the rates of focal adhesion assembly and disassembly were faster in Shot-depleted cells, leading to faster adhesion turnover that could underlie the increased migration speeds. This regulation of focal adhesion dynamics may be dependent on Shot being in an open confirmation. Using Drosophila border cells as an in vivo model for cell migration, we found that RNAi depletion led to precocious border cell migration. Collectively, these results suggest that spectraplakins not only function to cross-link the cytoskeleton but may regulate cell–matrix adhesion.

Interleukin-1 beta (IL-1β) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1β in cervical cancer cell proliferation and migration. HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1β overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT-PCR and Western blotting were used to test the expression of relevant genes. Exogenous IL-1β promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1β in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1β increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1β and the expression of JUN, RELB, and NF-κB2. Our data suggest that exogenous IL-1β regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1β, cervical tumor development, and cancer progression.

Current understandings on cell motility and directionality rely heavily on accumulated investigations of the adhesion-actin cytoskeleton-actomyosin contractility cycles, while microtubules have been understudied in this context. Durotaxis, the ability of cells to migrate up gradients of substrate stiffness, plays a critical part in development and disease. Here, we identify the pivotal role of Golgi microtubules in durotactic migration of single cells. Using high-throughput analysis of microtubule plus ends/focal adhesion interactions, we uncover that these non-centrosomal microtubules actively impart leading edge focal adhesion (FA) dynamics. Furthermore, we designed a new system where islands of higher stiffness were patterned within RGD peptide coated polyacrylamide gels. We revealed that the positioning of the Golgi apparatus is responsive to external mechanical cues and that the Golgi-nucleus axis aligns with the stiffness gradient in durotaxis. Together, our work unveils the cytoskeletal underpinning for single cell durotaxis. We propose a model in which the Golgi-nucleus axis serves both as a compass and as a steering wheel for durotactic migration, dictating cell directionality through the interaction between non-centrosomal microtubules and the FA dynamics.

Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. Here, we show that tyrosine phosphorylation of a focal adhesion protein, paxillin, crucially participates in these regulations. We found that tyrosine phosphorylation of paxillin was a prominent event upon integrin activation during epithelial-mesenchymal trans-differentiation and cell migration. Four major tyrosine phosphorylation sites were identified, and two of them were highly inducible upon integrin activation. Paxillin exhibits three distinct subcellular localizations as follows: localization along the cell periphery colocalized with circumferential actin meshworks, macroaggregation at focal adhesions connected to actin stress fibers, and diffuse cytoplasmic distribution. Tyrosine phosphorylation of paxillin localized at the cell periphery and focal adhesions was shown using phosphorylation site-specific antibodies. Mutations in the phosphorylation sites affected the peripheral localization of paxillin and paxillin-containing focal adhesion formation during cell migration and cell-cell collision, accompanied by altered actin organizations. Our analysis indicates that phosphorylation of multiple tyrosines in paxillin α is necessary for the proper function of paxillin and is involved in the temporospatial regulation of focal adhesion formation and actin cytoskeletal organization in motile cells.