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Vinculin binding to actin filaments is thought to be critical for force transduction within a cell, but direct experimental evidence to support this conclusion has been limited . In this study, we found mutation (R1049E) of the vinculin tail impairs its ability to bind F-actin, stimulate actin polymerization, and bundle F-actin in vitro. Further , mutant (R1049E) vinculin expressing cells are altered in cell migration, which is accompanied by changes in cell adhesion, cell spreading, and cell generation of traction forces, providing direct evidence for the critical role of vinculin in mechanotransduction at adhesion sites. Lastly, we herein discuss the viability of models detailing the F-actin-binding surface on vinculin in context of our mutational analysis.

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves.

Numerous events during development require the tightly controlled and regulated interaction of cells-from gastrulation in the early embryo to axonal pathfinding and remodeling of synaptic networks. Each of these events is dependent upon signals generated by cell-cell interactions, which are in turn specified by a diverse number of cell adhesion molecules. Many families of cell adhesion molecules have been described, and these fall into the broad categories of cadherins, immunoglobulin superfamily (IgSF) members, selectins, and integrins. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is a member of the IgSF, and controls numerous developmental processes, ranging from hematopoiesis to neuronal targeting. Furthermore, this protein has been implicated in the progression of numerous cancers of diverse origins. Despite the variety We certainly had many great times and made some unforgettable memories in the Biology Department. Eric Koehn, thank you for your logic, sound advice, and for your unlimited supply of bear hugs. Mark Lobas, you have been my 'partner in crime' in the Weiner Lab for more than four years. Thank you for always being there for me and v supporting me during the good, the bad, and the ugly times that I've encountered during my graduate career. I will miss seeing you in the lab every morning. Courtney Valerious, thank you for always being able to make me laugh, no matter the circumstances, for your constant encouragement, and for teaching me about life and weightlifting. Katie Dougherty, thank you for the "Come on! Get into it!" quote that I use to get myself fired up for difficult endeavors, and for reminding me to take breaks from work to have fun. Sarah Borwell, thank you for your friendship, for checking in on me, and for reminding me to not be too serious. Kaila Rome, you have always believed in me, even on my worst day, and I thank you for that. To the members of the Weiner Lab, both past and present-thank you for making this a wonderful place to work and learn over the past six years. Finally, to the Biology Department-Eileen, Tom, Misty, Phil, Bruce, Brenda, and others-all of you make this a wonderful place to work and learn. I cannot imagine having spent my graduate career anywhere else. vi TABLE OF CONTENTS

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