Dynamics Of Molecules And Membranes During The Early Stages Of Focal Adhesion Formation
Open Access
- Author:
- Fuentes, Daniela E.
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 13, 2012
- Committee Members:
- Peter J Butler, Dissertation Advisor/Co-Advisor
William O Hancock, Committee Member
Siyang Zheng, Committee Member
Sulin Zhang, Committee Member - Keywords:
- Mechanotransduction
endothelial cell
focal adhesion
membrane raft
adhesion - Abstract:
- Sensation and transduction of forces by endothelial cells are part of their inherent role as permeability barriers and regulators of blood flow in arteries, capillaries, and veins. Because of this proximity to blood flow, they are continuously exposed to mechanical forces that exhibit a wide range of spatial and temporal scales and directions. Among the key sensing mechanisms of the cell are focal adhesions (FA), and the plasma membrane. Dynamic and elusive in nature, membrane rafts have not been associated with being mechanosensitive, nor of being mechanically coupled to other mechanosensors. However, there is mounting evidence that focal adhesion formation and stability is sensitive to membrane composition. In order to test the hypothesis that membrane rafts participate in mechanosensation we developed a technique where single endothelial cells were deformed via FAs induced at the tip of a fibronectin (FN)-functionalized nanoelectrode probe. The dynamics of nascent FA assembly was assessed using time-lapsed fluorescent images of transfected RFP talin. We further hypothesized that membrane rafts, by virtue of their attachment to the cytoskeleton and high mobility in the plasma membrane, were mechanically coupled to FAs. To determine the kinetics and sequential order of raft and talin mechanosensitivity, time lapsed confocal fluorescent images were taken of cells during mechanical manipulation of a single induced FA by the nanoelectrode probe. Remote mechanosensing by rafts, sequential kinetics, and long term reversible accumulation was observed. These results demonstrate that rafts are directly mechanosensitive, mechanically coupled to focal adhesions, and that raft mobility may enable the earliest events related to FA mechanosensing. To better understand adhesion and the role of membrane composition, an optical trap was used to manipulate a FN functionalized bead near a cell in order to monitor dynamic adhesion events and particle tracking of DIC images was used to assess surface fluctuations and adhesion. Cholesterol depletion, which disrupts membrane rafts, reduced adhesion time, suggesting a role of the membrane in adhesion. Additionally, an increase in effective diffusion of functionalized beads upon early contact points to the importance of membrane fluctuations in adhesion. Together, these findings provide us with insight to a new mechanosensor, the membrane raft, as well as a more holistic understanding of adhesion that involves adhesion proteins, their dynamics, and the role of the plasma membrane in their transport and accessibility.