Investigators: Prof. C.F.Dewey, MIT
Prof. J.H.Hartwig, Brigham and Women's Hospital, Harvard Medical School
Arterial endothelial cells perform a crucial role in maintaining vascular integrity. Their dynamics and biochemical activity influence arterial muscle tone and control fluid and nutrient transport between the blood and the artery wall. They also exhibit cell division and motility to repair wounds, and modulate receptors that determine cellular and molecular attachment. The behavior of endothelial cells is profoundly affected by their internal structure, which is characterized by a dynamic actin meshwork. The overall goal of this work is to understand the actin dynamics in endothelial cells and the changes that occur when the cells are subjected to fluid shear stresses.
|The primary actin structure in endothelial cells is a three dimensional orthogonal network of actin that interconnects the more basal stress fibers. During accommodation to fluid flow, actin stress fibers become more prominent and align with the direction of fluid flow. Surprisingly, relatively minor changes are found into the orthogonal actin gel that fills the bulk of the cytoplasmic space suggesting that this network either mechanically resists the applied shear stress or, if deformed, is dynamically remodeled back to a preferred architecture. 3-D actin organization is generated and maintained by proteins that bind and crosslink actin filaments into rigid networks. While many proteins have been identified and characterized with respect to their actin crosslinking properties, two molecules are of particular importance to the actin structures of endothelial cells: filamin and a-actinin. Filamin is a family of elongated molecules concerned with the creation and stabilization of orthogonal actin networks. a-Actinin is an actin crosslinker that aligns filaments into stress fiber bundles.|
The cell cytoskelton forms a 3-D structural network that gives the cytoplasm its shape, form, and mechanical properties. We have developed vision-based method that automatically recontructs its 3-D structure from paired micrographs taken at the different tilt angles in the electron microscope.
As more images become available, the reconstruction is refined. And also, tomography-based reconstruction can be applied. The following movie clip shows an example of recontruction with about 60 images. (You need QuickTime Player or Window MediaPlayer to see it.)
|Our studies reveal that the maximal actin filament content and slowest actin fiber turnover rates occur in confluent, immobile endothelial cells. Filament content approaches 70% of the total in cells comprising a quiescent monolayer and filament turnover half-times are on the order of 40 min. Removal of contact inhibition stimulates endothelial cells to crawl, resulting in a dramatic decrease in their cytoplasmic filament content to ~40% of the total and acceleration of actin filament turnover by 5 to 10 fold. The left figure summarizes the activity of important regulatory proteins with regard to actin filament turnover in cells.|
The fluorescence methods have been developed and
successfully implemented to measure the cytoskeletal dynamics of endothelial cells. The
system allows simultaneous measurement of the actin monomer diffusion rate, the
monomer/polymer ratio, and the turnover rate of polymer filaments and has been applied in
several different cell types and dynamic states (free cells and cells in a monolayer). The
theory has been successfully applied to both photoactivated fluorescence (PAF) and
fluorescent recovery after photobleaching (FRAP). We have correlated the rate of actin
filament turnover to motility in individual endothelial cells. First, this establishes
baseline values for filament turnover, filament content, and monomer diffusion for all
current and future work. Second, it demonstrates that the rate of filament turnover is
altered by individual cells when they change their rate of movement. Third, we measure a
striking inverse relationship between actin filament content and speed of cell movement.
Another critical finding is the recent discovery of multiple filamin family members and the generation of reagents to dissect the role of each protein in endothelial cell function. These findings suggest that filamin-2 and 3 will interact with unique and important cytoplasmic and membrane proteins.
The hemodynamic environment of the vasculature is a potent regulator of endothelial cell structure and function. Endothelial cells in vivo differ in pathophysiology depending on their location in the arterial tree and the shear stress gradients to which they are exposed. Laminar, steady flow over endothelial cells for 24 hours at 10-12 dynes/cm2 has been previously shown to alter cell shape from a polygonal profile to aligned and elongated in the direction of flow. Areas of arterial bifurcations and flow reversal, most prone to development of atherosclerotic plaques, have been prototyped in vitro with the use of disturbed flow simulations. These studies reveal lack of flow orientation, increased cellular differentiation, and binding of leukocytes. Numerous investigations have been performed to determine the consequences of this transition from static to dynamic culture. Endothelial cells in response to flow are known to alter gene expression, transiently increase cytosolic calcium, release vasoactive substances, increase levels of inositol phosphates, exhibit heightened permeability, and augment existing focal adhesion site and associated anchoring proteins, in an a dramatic sequence of events resulting in reorganization of the underlying actin filament structure.
We are currently examining the role of fluid shear stress in modulating the actin dynamics of human and bovine endothelial cells. Experiments focus on determining the temporal relation between cellular morphology and the mechanical cytoskeletal remodeling parameters obtained from PAF in endothelial cells under fluid flow.
Recent advances in NMR imaging with high magnetic fields has made it possible to study the diffusion coefficient of molecules in solution. We are initiating a program with Prof. David Cory of MIT to make independent measurements of the actin diffusion coefficient in endothelial cells. This program is still in its early stages, and no definitive results are available at the present time.