| Cells display dramatic differences in morphology when they are in 3D matrices vs. on 2D substrata. The structural mechanisms underlying these differences are not understood. MTs are known to determine the polarity of protrusions and to influence cell shape in many cell types. This dissertation uses morphometric analysis to identify key morphological differences between cells in 2D vs. 3D, and tests the role of microtubules (MTs) in determining those differences. Bovine aortic endothelial cells (BAECs) cultured on 2D substrata and in 3D collagen gels were examined by time-lapse imaging, immuno-fluorescence, and confocal microscopy. In 3D matrices BAECs formed cylindrical branching pseudopodia, but formed wide, flat lamellae in 2D. Quantitative analysis of motile behavior and cytoskeletal organization revealed three distinct cytoplasmic zones in both cases: (i) A small, F-actin rich, rapidly moving peripheral zone, (ii) a larger, more stable, intermediate zone characterized by abundant MTs, and (iii) a locomotively inert central zone. To determine if MTs control the organization of these zones in 3D vs. 2D, MTs were depolymerized with Colcemid. This reduced the peripheral zone and the intermediate zone of BAECs on glass, and completely prevented extension of the intermediate zone of BAECs in 3D. Time-lapse images showed that Colcemid-treated BAECs formed small protrusions and pulled on the collagen matrix in 3D, and immuno-cytochemistry revealed accumulation of the focal-adhesion protein, vinculin, at the cell periphery, suggesting that MTs play a role in regulating cell adhesion/traction. It was hypothesized that MTs are necessary for balancing contractile and protrusive forces. To test this, BAECs were cultured on more rigid collagen surfaces, increasing resistance to contraction, or inside collagen gels in the presence of a Rho-kinase inhibitor, Y-27632, known to inhibit myosin-based contractility. In both cases, extension of the intermediate zone occurred even in the absence of MTs. This works shows that MTs play an important role in regulating traction forces. This control is especially important for cells in 3D matrices like those found in vivo, because excessive force may break the surrounding lattice and prevent cell extension, and insufficient force would fail to move the cell. |
