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Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

The major impact of this research lies in the aspect of improved design and long term biocompatibility of materials used for implants. There are two goals in this research. The first goal is to develop a methodology to quantitatively measure cell-material adhesion. The second goal is to obtain fundamental understanding of cell-material adhesion mechanisms. A rotating parallel disk is used to measure cell adhesion. The rotational system applies a controlled shear stress to the cultured cells. The shear stress experienced by the cells varies with radial location, being highest at the edge and zero at the disk?s center. There is a critical point along the radius where the shear stress experienced by the cells equals their adhesion strength. The cells outside it are removed and the cells inside it remain attached to the surface. NIH 3T3 Swiss mouse fibroblasts and chick retina neuron cells from 6-day embryos are used in this study. The fibroblasts were cultured on poly(methyl methacrylate) (PMMA), polycarbonate (PC), and on gold coated poly(vinylidene fluoride) (Au/PVDF). The critical shear stress for fibroblasts was the lowest for PC with 5.09 dynes/cm2 and highest for PMMA with 21.0 dynes/cm2. This four-fold difference is mainly due to the chemical structure of PMMA which promotes higher cell adhesion when compared to PC. Neurons were cultured on poly-D-lysine coated glass to promote cell adhesion. The critical shear stress of neuron cells varied from 3.94 to 27.8 dynes/cm2 these values are directly proportional to the applied shear stress. The neuron adhesion plateau at ~27 dynes/cm2 which indicates the maximum adhesion strength of the neuron/poly-D-lysine coated glass pair. This thesis contains six chapters. Chapter I describes the importance of cell adhesion for biocompatibility. Chapter II describes in more detail the goals of this research and the expected results. Chapter III lists all the materials, equipment, and methods used in this study. The most significant results are summarized in Chapter IV. The observations and results obtained are explained in detail in Chapter V and Chapter VI describes the key outcomes as well as proposes questions for the advancement of this research.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Biomedical Engineering." Includes bibliographical references (p. 79-81).

Current biomaterials are designed to be passive in nature to prevent the initiation of adverse immune responses upon contact with biological substances. While this approach of inertness is still a crucial design component for some applications, the possibility of engineering desired cell responses in the local environment of the material exists and is of particular interest in implantable devices and tissue engineered constructs. Fundamental knowledge of the relationships between cell adhesion and gross cell behavior will provide key design criteria for the creation of advanced biomaterials that induced locally controlled cellular responses. This work investigates the possibility of engineering cell behavior by limiting adhesion site maturation. Chemically-defined nanoislands of fibronectin were created using a combination of nanosphere lithography and an orthogonal surface functionalization strategy. Investigation of the adhesive and cytoskeletal components of cells cultured on these surfaces demonstrates that chemically-defined nanopatterns provide an upper size limit to adhesion site growth which in turn influences the degree of cytoskeletal formation. The imposed restriction on adhesion site growth results in the formation of a relatively higher number of more evenly distributed, small adhesions throughout the cell body. The adhesive behavior can be tuned by changing the nanopattern properties with respect to their size, spacing, and density. Furthermore, it is demonstrated that the observed differences in cell adhesion as imposed by the nanopatterned surfaces induces changes in gross cell behavior with respect to spreading, proliferation, and motility. The results presented here parallel observations documented in cells cultured on elastic surfaces and indicate that intracellular signaling cascades initiated and governed by cellular adhesion sites are sensitive to adhesion size/maturation and possibly the amount of force generated locally at these adhesion sites. The conclusions drawn from these studies give insight into the possibility of implementing nanostructured biomaterials for cell engineering purposes and provide design criteria for the next generation of tissue engineered constructs. / text