SHEAR STRESS ELICITS A TRANSIENT DOMAIN-DEPENDENT ALTERATION OF THE PLASMA MEMBRANE FLUIDITY IN ENDOTHELIAL CELLS
Open Access
- Author:
- Tabouillot, Tristan
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 26, 2009
- Committee Members:
- Peter J Butler, Dissertation Advisor/Co-Advisor
Peter J Butler, Committee Chair/Co-Chair
Ahmed A Heikal, Committee Member
Erin Elizabeth Sheets, Committee Member
Herbert Herling Lipowsky, Committee Member - Keywords:
- mechanotransduction
endothelial
fluorescence lifetime
fluorescence correlation spectroscopy
plasma membrane - Abstract:
- Blood flow-associated shear stress causes physiological and pathophysiological biochemical processes in endothelial cells that may be initiated by alterations in plasma membrane domains. Time scales of cells signaling cascades triggered by shear stress spans seconds to hours. Hence, a single-molecule fluorescence spectroscopy system was developed to investigate cellular molecular dynamics with high spatial and temporal resolution. We defined a procedure to locate lipoid dyes in cultured endothelial cells under physiological conditions. Thus, we assessed the photophysics and dynamics of the lipid analogues DiI-C12 and DiI-C18 in endothelial cells subjected to physiological fluid shear stress. These lipoid dyes have been proposed to segregate to domains of the membrane with thicknesses matching DiI alkyl chain length such that DiI-C12 partitions into the thinner liquid-disordered phase and DiI-C18 partitions into the thicker liquid-ordered phase. DiI fluorescence lifetime, molecular brightness, number of molecules, and lateral diffusion were obtained from time-correlated single photon counting data and analyzed as a function of shear stress and time. DiI-C12 fluorescence lifetime was 0.16 ns faster than DiI-C18 lifetime and the diffusion coefficient of DiI-C12 was 1.5 times greater than the DiI-C18 diffusion coefficient confirming that DiI-C12 probes more fluid membrane environments as compared to DiI-C18. Domains probed by DiI-C12 dye exhibited a decrease of fluorescence lifetime from 5 to 20 seconds after the onset of shear while domains probed by DiI-C18 exhibited a decrease of fluorescence lifetime that was delayed 10 seconds but was sustained for the 2 minutes the cells were subjected to flow. Determination of the number of fluorophores in the observation volume from moment analysis suggested that DiI-C12 -labeled domains increased in area while DiI-C18 -stained membrane did not. Taken together, the data suggest that shear stress induces an early rippling of DiI-C12 –stained domains and later change in lipid mobility of DiI-C18 –stained domains. These results help focus attention on the membrane microdomains interaction with glycocalyx and cytoskeleton as sites of shear sensitivity.