The Golgi microtubules regulate single cell durotaxis.

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.

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.

Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands

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.

Adhesion-based migration is regulated by focal adhesions, multi-protein nanostructures linking the intracellular cytoskeleton to the extracellular substrate. Efficient adhesion-based migration has been shown to be regulated by focal adhesion dynamics such as lifetime, size and turnover, which in turn are influenced by the molecular composition of focal adhesions. We recently identified the formation of cell-substrate adhesion populations in Dictyostelium discoideum with differing molecular compositions, but it is unclear how these distinct compositions influence Dictyostelium adhesion dynamics and cell migration. Here, we further investigate the role of molecular composition on Dictyostelium adhesion lifetime and protein turnover during cell migration. We show that co-localization of VinculinB to PaxillinB-positive cell-substrate adhesions increases adhesion lifetime without changing PaxillinB turnover. We further show that perturbing cell-substrate adhesion composition with a PaxillinB N-terminal truncation increases adhesion lifetime and decreases PaxillinB turnover at adhesions. These findings suggest that similar to mammalian focal adhesions, molecular composition of Dictyostelium cell-substrate adhesion regulates their adhesion lifetimes and protein turnover, providing insight into how cell-substate adhesions function during Dictyostelium cell migration.

The human cytomegalovirus (HCMV) US12 gene family contributes to virus-host interactions by regulating the virus' cell tropism and its evasion of host innate immune responses. US21, one of the 10 US12 genes (US12-US21), is a descendant of a captured cellular transmembrane BAX inhibitor motif-containing gene. It encodes a 7TMD endoplasmic reticulum (ER)-resident viroporin (pUS21) capable of reducing the Ca2+ content of ER stores, which, in turn, protects cells against apoptosis. Since regulation of Ca2+ homeostasis affects a broad range of cellular responses, including cell motility, we investigated whether pUS21 might also interfere with this cytobiological consequence of Ca2+ signaling. Indeed, deletion of the US21 gene impaired the ability of HCMV-infected cells to migrate, whereas expression of US21 protein stimulated cell migration and adhesion, as well as focal adhesion (FA) dynamics, in a way that depended on its ability to manipulate ER Ca2+ content. Mechanistic studies revealed pUS21-mediated cell migration to involve calpain 2 activation since its inhibition prevented the viroporin's effects on cell motility. Pertinently, pUS21 expression stimulated a store-operated Ca2+ entry (SOCE) mechanism that may determine the activation of calpain 2 by promoting Ca2+ entry. Furthermore, pUS21 was observed to interact with talin-1, a calpain 2 substrate, and crucial protein component of FA complexes. A functional consequence of this interaction was confirmed by talin-1 knockdown, which abrogated the pUS21-mediated increase in cell migration. Together, these results indicate the US21-encoded viroporin to be a viral regulator of cell adhesion and migration in the context of HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic pathogen that owes part of its success to the capture, duplication, and tuning of cellular genes to generate modern viral proteins which promote infection and persistence in the host by interfering with many cell biochemical and physiological pathways. The US21 viral protein provides an example of this evolutionary strategy: it is a cellular-derived calcium channel that manipulates intracellular calcium homeostasis to confer edges to HCMV replication. Here, we report on the characterization of a novel function of the US21 protein as a viral regulator of cell migration and adhesion through mechanisms involving its calcium channel activity. Characterization of HCMV multifunctional regulatory proteins, like US21, supports the better understanding of viral pathogenesis and may open avenues for the design of new antiviral strategies that exploit their functions.

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.

Background:The dynamics of focal adhesion (FA) turnover is a key determinant for the regulation of cancer cell migration. Here we investigated FA turnover in a panel of breast cancer models with distinct invasive properties and evaluated the impact of reversine on this turnover in relation to cancer cell invasion in in vitro and in vivo conditions.Methods:Live imaging and immunofluorescence assays were used to investigate FA turnover in breast cancer cells. Biochemical studies were used to investigate the impact of reversine on FA signalling and turnover. In vivo activity was investigated using orthotopic breast cancer mouse models.Results:Accelerated FA disassembly from plasma membrane protrusions was observed in invasive compared with non-invasive breast cancer cells or non-immortalised mammary epithelial cells. Reversine significantly inhibited FA disassembly leading to stable FAs, which was associated with reduced cell motility and invasion. The inhibitory effect of reversine on FA turnover accounted for a large part on its capacity to interfere with FAK function on regulating its downstream targets. In orthotopic breast cancer mouse models, reversine revealed a potent inhibitory activity on tumour progression to metastasis.Conclusion:These results support the utility of targeting FA turnover as a therapeutic approach for invasive breast cancer.

Paxillin is a well-known multidomain scaffold protein that is involved in the regulation of cell-matrix adhesiondynamics, a process required for the tumor cell migration and invasion. Phosphorylation of the serine residue 178requires c-Jun NH2-terminal kinase (JNK) activation, which occurs downstream of epidermal growth factor receptor (EGFR)-mediated signaling and drives cell migration. In this study, we investigated the significance of paxillin Ser178 phosphorylation in breast cancer progression.We employed the rat mammary carcinoma MTLn3 cell line with which we established stabile variants of both wild type and mutant GFP-paxillin constructs. With those, we next performed several in vitro assays including cell proliferation, migration and focal adhesion dynamics. Finally, we monitored the metastatic spread of both cell line variants in an othrotopic mouse model for breast cancer.Here we show that expression of the phospho-defective mutant paxillinS178A in the metastatic mammary adenocarcinoma MTLn3 cell-line significantly decreased EGF-induced cell migration, which was correlated with impaired focal adhesion dynamics. Moreover, paxillinS178A attenuated lung metastasis formation in an orthotopic in vivo mammary gland tumor/metastasis model, demonstrating the importance of JNK-mediated paxillin phosphorylation in breast cancer progression. Expression of paxillinS178A caused a decrease in EGFR expression while re-expression of EGFR in MTLn3-paxillinS178A cells fully restored EGF-driven cell motility and focal adhesion dynamics. Furthermore, re-expression of EGFR in MTLn3-paxillinS178A rescued spontaneous metastasis from breast to lung.Overall our data show an important role for JNK-mediated paxillin Ser178 phosphorylation in the regulation of EGFR expression and thereby, in EGF-driven cell migration and metastasis formation.

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.

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.

Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

The migration of tumor cells of different degrees of invasivity is studied, on the basis of the traction forces exerted in time on soft substrates (Young modulus∼10 kPa). It is found that the outliers of the traction stresses can be an effective indicator to distinguish cancer cell lines of different invasiveness. Here, we test two different epithelial bladder cancer cell lines, one invasive (T24), and a less invasive one (RT112). Invasive cancer cells move in a nearly periodic motion, with peaks in velocity corresponding to higher traction forces exerted on the substrate, whereas less invasive cells develop traction stresses almost constant in time. The dynamics of focal adhesions (FAs) as well as cytoskeleton features reveals that different mechanisms are activated to migrate: T24 cells show an interconnected cytoskeleton linked to mature adhesion sites, leading to small traction stresses, whereas less invasive cells (RT112) show a less-structured cytoskeleton and unmature adhesions corresponding to higher traction stresses. Migration velocities are smaller in the case of less invasive cells. The mean squared displacement shows super-diffusive motion in both cases with higher exponent for the more invasive cancer cells. Further correlations between traction forces and the actin cytoskeleton reveal an unexpected pattern of a large actin rim at the RT112 cell edge where higher forces are colocalized, whereas a more usual cytoskeleton structure with stress fibers and FAs are found for T24 cancer cells. We conjecture that this kind of analysis can be useful to classify cancer cell invasiveness.

Focal adhesions (FAs) are important adhesion sites between eukaryotic cells and the extracellular matrix: They mediate cell adhesion, spreading and motility. Over the last decade it has become evident that FAs are bi-directional mechano-chemical devices: They both exert and sense physical forces by converting biochemical signals into mechanical force and vice versa. As such, they represent a highly fascinating interface between physics and biology. Recently, it has been shown that the intrinsic force generated by the contractile machinery of the cell that leads to FA growth can be substituted by external forces. However, the exact mechanism behind FA-mediated mechanosensing remained unclear. This unsolved question has stimulated several competing theories that attempt to model the physical principles governing the force-induced assembly of adhesion plaque proteins. In this thesis, we present a novel, inexpensive method to micromanipulate living cells with single elastic micropillars and discuss the effect of lateral shear stress on focal adhesion dynamics of fibroblasts. We have successfully induced both growth and disassembly of FAs by shearing cells. Dynamics of single FAs and intensity profiles along their major axes have been analyzed in detail. We find distinct features for stretched respectively relaxed FAs. The presented data will be valuable for the further refinement, verification or falsification of theories in this field.