Nano-Stenciled RGD-Gold Patterns That Inhibit Focal Contact Maturation Induce Lamellipodia Formation in Fibroblasts

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 µm. The gold was functionalized with RGD peptide as ligand for cellular integrins, and mouse embryo fibroblasts were plated. Limiting the length of cell-matrix adhesions to 500 nm or less disturbed the maturation of vinculin-positive focal complexes into focal contacts and fibrillar adhesions, as indicated by poor recruitment of α5-integrin. We found that on sub-micrometer patterns, fibroblasts spread extensively, but did not polarize. Instead, they formed excessive numbers of lamellipodia and a fine actin meshwork without stress fibers. Moreover, these cells showed aberrant fibronectin fibrillogenesis, and their speed of directed migration was reduced significantly compared to fibroblasts on 2 µm square patterns. Interference with RhoA/ROCK signaling eliminated the pattern-dependent differences in cell morphology. Our results indicate that manipulating the maturation of cell-matrix adhesions by nanopatterned surfaces allows to influence morphology, actin dynamics, migration and ECM assembly of adhering fibroblasts

Loss of the integrin-activating transmembrane protein Fam38A (Piezo1) promotes a switch to a reduced integrin-dependent mode of cell migration

Lung cancer is one of the most common fatal diseases in the developed world. The disease is rarely cured by currently available therapies, with an overall survival rate of ∼10%. Characterizing novel proteins that offer crucial insights into the processes of lung tumour invasion and metastasis may therefore provide much-needed prognostic markers, and influence therapeutic strategies. Aberrant function of the integrin family of heterodimeric cell surface receptors is a common theme in cancer--investigation into novel integrin activity regulators may offer crucial insights into the processes of tumour invasion and metastasis and may reveal insights into potential therapeutic targets. We previously described that depletion of the novel multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER), inactivates endogenous beta1 integrin affinity, reducing cell adhesion. We now show that depletion of Fam38A, also now known as Piezo1, causes anchorage independence and a switch to a reduced integrin-dependent mode of cell migration/invasion, a novel phenotype for this integrin-regulating protein. Normal lung epithelial cells show increased rates of migration by 2D time-lapse microscopy and increased capacity to invade into matrigel, despite having decreased integrin affinity. We confirm greatly depleted Fam38A expression in small cell lung cancer (SCLC) lines where a form of reduced integrin-dependent migration, i.e. amoeboid migration, is a known phenotype. We propose that loss of Fam38A expression may cause increased cell migration and metastasis in lung tumours

Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension

Cytosolic SYT/SS18 Isoforms Are Actin-Associated Proteins that Function in Matrix-Specific Adhesion

SYT (SYnovial sarcoma Translocated gene or SS18) is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices

Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA

Interplay between cell adhesion and growth factor receptors: from the plasma membrane to the endosomes

The emergence of multicellular animals could only take place once evolution had produced molecular mechanisms for cell adhesion and communication. Today, all metazoans express integrin-type adhesion receptors and receptors for growth factors. Integrins recognize extracellular matrix proteins and respective receptors on other cells and, following ligand binding, can activate the same cellular signaling pathways that are regulated by growth factor receptors. Recent reports have indicated that the two receptor systems also collaborate in many other ways. Here, we review the present information concerning the role of integrins as assisting growth factor receptors and the interplay between the receptors in cell signaling and in the orchestration of receptor recycling

α5β1 Integrin-Mediated Adhesion to Fibronectin Is Required for Axis Elongation and Somitogenesis in Mice

The arginine-glycine-aspartate (RGD) motif in fibronectin (FN) represents the major binding site for α5β1 and αvβ3 integrins. Mice lacking a functional RGD motif in FN (FNRGE/RGE) or α5 integrin develop identical phenotypes characterized by embryonic lethality and a severely shortened posterior trunk with kinked neural tubes. Here we show that the FNRGE/RGE embryos arrest both segmentation and axis elongation. The arrest is evident at about E9.0, corresponding to a stage when gastrulation ceases and the tail bud-derived presomitic mesoderm (PSM) induces α5 integrin expression and assumes axis elongation. At this stage cells of the posterior part of the PSM in wild type embryos are tightly coordinated, express somitic oscillator and cyclic genes required for segmentation, and form a tapered tail bud that extends caudally. In contrast, the posterior PSM cells in FNRGE/RGE embryos lost their tight associations, formed a blunt tail bud unable to extend the body axis, failed to induce the synchronised expression of Notch1 and cyclic genes and cease the formation of new somites. Mechanistically, the interaction of PSM cells with the RGD motif of FN is required for dynamic formation of lamellipodia allowing motility and cell-cell contact formation, as these processes fail when wild type PSM cells are seeded into a FN matrix derived from FNRGE/RGE fibroblasts. Thus, α5β1-mediated adhesion to FN in the PSM regulates the dynamics of membrane protrusions and cell-to-cell communication essential for elongation and segmentation of the body axis

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration