Petroll, W. Matthew2010-07-122010-07-122008-05-132008-05-13https://hdl.handle.net/2152.5/562The overall goal is to develop and apply quantification techniques for assessing the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions in corneal fibroblasts at the sub-cellular level. To specifically study how Rho and Rac regulate sub-cellular mechanical behavior, cells were plated inside 3-D matrices and incubated with activators and/or inhibitors of Rho and Rac and 3-D optical section images were collected simultaneously. Cell morphology, collagen density and orientation were quantitatively studied. The first important finding is that Rho kinase-dependent contractile force generation leads to co-alignment of cells. This process contributes to global matrix contraction and thus may play a central role in cell transformation and force generation during wound healing. In contrast, activation of Rac using PDGF induces dramatic cell elongation without significant matrix reorganization. PDGF may play a role in cell migration during wound healing process since migration requires protrusion of cell extensions, but not necessarily large contractile force. In order to obtain more detailed understanding of how cells reorganize matrices over time, 4-D imaging techniques were used. Cells were plated inside 3-D matrices and time-lapse DIC and LSCM imaging was performed while disrupting cytoskeletal proteins in the presence or absence of the Rho kinase inhibitor. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture. Taken together, these experiments demonstrate that quantitative static and dynamic imaging of cells in 3-D matrices is capable of providing unique insights into the role of specific signaling pathways on the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions.Electronicapplication/pdfenCorneal KeratocytesMicrotubulesrho-Associated KinasesFibroblastsCell ShapeThesisborn digital422595791