CK2ß Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2ß regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2ß compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2ß in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2ß loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2ß as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

Immunofluorescence of Cell-Cell and Cell-Extracellular Matrix Adhesive Defects in In Vitro Endothelial CCM Model: Juxtacrine Role of Mutant Extracellular Matrix on Wild-Type Endothelial Cells.

Endothelial cells lining cerebral cavernous malformations (CCM) present strong adhesive and mechanical defects. Increased cell contractility is a driver to the onset and the expansion of the CCM lesions. 2D in vitro endothelial models have been developed from either endothelial cells isolated from ccm1-3 knock-out mice or CCM1-3-silenced primary endothelial cells. These in vitro models faithfully recapitulate the adhesive and contractile defects of the CCM-deficient endothelial cells such as increased cell-extracellular matrix (ECM) adhesion through ß1 integrin-anchored actin stress fibers, abnormal remodeling of the ECM, and destabilized VE-cadherin-dependent cell-cell junctions. Using such 2D in vitro CCM models, we have shown that the ECM remodeled by CCM-depleted endothelial cells can propagate CCM-like adhesive defects to wild-type endothelial cells, a process potentially pertinent to CCM lesion expansion. Here, we detail methods for studying the morphology of focal adhesions, actomyosin cytoskeleton, and VE-cadherin-dependent Adherens junctions by immunofluorescence and morphometric analyses. Moreover, we detail the protocols to produce and purify remodeled ECM and to test its effect on endothelial cell adhesion.

Microenvironment, tumor cell plasticity, and cancer.

PURPOSE OF REVIEW: Much effort has been devoted to determining how cellular and noncellular components of the tumoral niche initiate and promote cancer development. Cancer cells perceive biochemical signals from components of the extracellular matrix (ECM) and sense physical features, such as matrix stiffness and cell confinement. The past decade has seen a better understanding of the biophysics and mechanobiology associated with cancer cells. Indeed, loss of mechanisms controlling the production, the degradation, and the remodeling of ECM contributes to tumor growth or cell dissemination by affecting cell contractility in response to ECM stiffness and by stimulating mechanical dependence of growth factor activation. RESULTS: Cell plasticity allows adaptative strategies for cancer cells to survive or eventually escape from tumoral environment through modification of the microenvironment-cell interface, internal tension increase, and nuclear deformation partly leading to intratumoral heterogeneity. However, although alteration of the biomechanical properties of the ECM are sufficient to promote cell migration and invasion in cancer cells, this microenvironment can also provide a hospitable niche for tumor dormancy and resistance to cancer therapy. CONCLUSION: The review will focus on how physicochemical properties of ECM might promote tumor growth or cell dissemination or on the contrary maintain quiescent state of cancer cells. It is crucial to clarify the molecular basis of mechanotransduction in the development and progression of tumors to identify new potential biomarkers and anticancer therapeutic targets.

CCM1-ICAP-1 complex controls ß1 integrin-dependent endothelial contractility and fibronectin remodeling.

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of ß1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased ß1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1-deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of ß1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.

Podosome-type adhesions and focal adhesions, so alike yet so different.

Cell-matrix adhesions are essential for cell migration, tissue organization and differentiation, therefore playing central roles in embryonic development, remodeling and homeostasis of tissues and organs. Matrix adhesion-dependent signals cooperate with other pathways to regulate biological functions such as cell survival, cell proliferation, wound healing, and tumorigenesis. Cell migration and invasion are integrated processes requiring the continuous, coordinated assembly and disassembly of integrin-mediated adhesions. An understanding of how integrins regulate cell migration and invasiveness through the dynamic regulation of adhesions is fundamental to both physiological and pathological situations. A variety of cell-matrix adhesions has been identified, namely, focal complexes, focal adhesions, fibrillar adhesions, podosomes, and invadopodia (podosome-type adhesions). These adhesion sites contain integrin clusters able to develop specialized structures, which are different in their architecture and dynamics although they share almost the same proteins. Here we compare recent advances and developments in the elucidation of the organization and dynamics of focal adhesions and podosome-type adhesions, in order to understand how such subcellular sites - though closely related in their composition - can be structurally and functionally different. The underlying question is how their respective physiological or pathological roles are related to their distinct organization.

A deconvolution method for the separation of specific versus nonspecific interactions in noncovalent protein-ligand complexes analyzed by ESI-FT-ICR mass spectrometry.

A method to separate specific and nonspecific noncovalent interactions observed in ESI mass spectra between a protein and its ligands is presented. Assuming noncooperative binding, the specific ligand binding is modeled as a statistical distribution on identical binding sites. For the nonspecific fraction we assume a statistical distribution on a large number of "nonspecific" interacting sites. The model was successfully applied to the noncovalent interaction between the protein creatine kinase (CK) and its ligands adenosine diphosphate (ADP) and adenosine triphosphate (ATP) that both exhibit nonspecific binding in the mass spectrum. The two sequential dissociation constants obtained by applying our method are K(1,diss) = 11.8 +/- 1.5 microM and K(2,diss) = 48 +/- 6 microM for ADP. For ATP, the constants are K(1,diss) = 27 +/- 7 microM and K(2,diss) = 114 +/- 27 microM. All constants are in good correlation with reported literature values. The model should be valuable for systems with a large dissociation constant that require high ligand concentrations and thus have increased potential of forming nonspecific adducts.