Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 132-157). Also available online through Digital Dissertations.

Focal adhesions are dynamic multiprotein structures connecting cells to their surrounding microenvironment. Cells receive critical mechanical signals from adhesions that control many cellular processes including wound healing, differentiation, development, and cancer. Proteins that form adhesions are called adhesion proteins and some of these proteins can be mechanosensitive, meaning that they respond to mechanical stimuli. During spreading and migration, cells mechanically test extracellular matrix rigidity by contracting matrix to a constant displacement. Transmission and processing of such mechanical signals rely upon the dynamic regulation of the adhesions, which is tightly coordinated with activation of intracellular signaling cascades involving various adhesion molecules. However, the molecular mechanisms of mechanical signals that are transmitted through the adhesions to control cell behavior are poorly understood. In this thesis, we discovered novel phenomenon and mechanisms to elucidate roles of mechanical signals for multiple key aspects of basic cell behavior, especially cell growth. We performed live cell imaging of cells spreading on fibronectin coated micropillars to understand adhesion formation, adhesion regulation, and their impact on cell behavior. One of the earliest molecules to arrive at an adhesion formation site is a mechanosensitive protein called talin which binds to several other entities to form the backbone of focal adhesions. We found a novel role of talin cleavage, which previously was thought to play a role only in focal adhesion turnover. We found that talin cleavage is a force dependent process that regulates proper adhesion formation, thereby governing several critical cellular processes. In the absence of this talin cleavage, cells formed abnormal adhesions and showed inhibited growth. Further, we found that upon inhibition of talin cleavage, one of the key cellular behaviors of increased cellular motility upon stimulation by epidermal growth factor seemed to disappear. Epidermal growth factor receptor is a transmembrane protein and has previously been shown to play important role in various cancers where cells exhibit altered rigidity sensing. Surprisingly, we found that epidermal growth factor receptor was required for cellular rigidity sensing only on rigid substrates, highlighting the importance of the interplay between mechanical and biochemical signals in determining cell behavior.

Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.

The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

The protein p130Cas (human ortholog BCAR1) is a major substrate for phosphorylation by the Src family kinase and plays a central role in oncogenic transformation. Increased level of BCAR1 correlates with primary tumour growth and cancer progression. Localized to focal adhesion, p130Cas serves as a mechanosensor and mediates key interactions with the extracellular environment. The structure of p130Cas is crucial for its function, mainly the anchoring domains SH3 and CCH, together with the substrate domain which is extended when under tension. This Master's thesis presents a newly developer FRET mechanosensor based on the structure of p130Cas. The sensor utilizes the anchoring domains of p130Cas for proper localization to focal adhesions, where it can detect tension in living cells. Key words: p130CAS, FRET, focal adhesions, mechanosensing

Focal adhesions are important subcellular structures that are composed of many signaling and scaffolding proteins. They serve not only for anchoring the cell to the substratum but they are also important signaling centers that regulate various cellular behavior such as migration, invasiveness, proliferation and survival. Focal adhesion signaling needs to be strictly regulated because alteration in activity or expression of many focal adhesion proteins leads to tumorogenesis and metastasis formation. One of the most important scaffolding protein associated with focal adhesion is p130Cas. The importance of p130Cas in regulation of cell migration and invasiveness has been well established. P130Cas also plays important role in regulation of cell survival and proliferation. Moreover, high protein levels of human ortholog of p130Cas - BCAR1, has been linked to more aggressive breast tumors and poor prognosis. During my doctoral studies, I focused on the role of p130Cas in integrin signaling. At the beginning we characterized the role of tyrosine 12 phosphorylation within its SH3 domain. We confirmed that this phosphorylation is increased in Src527F transformed mouse embryonic fibroblasts compared to non-transformed counterparts and also in some human cancer cell lines. We showed that this phosphorylation...

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2001. / Spine title: Yersinia uptake by mammalian cells. Includes bibliographical references (leaves 214-240). Also available online through Digital Dissertations.

Proteins represent major constituent of the extracellular matrix which plays an important role in the formation, maintenance and remodeling of tissues, this project focuses on adsorption of two specific serum proteins fibronectin (FN) and vitronectin (VTN) responsible for mediating cell matrix interaction through integrin binding, tripeptide Arg-Gly-Asp (RGD) sequence found in these protein features are recognized by αβV3 integrin which ultimately helps in clot formation.