Ravi K. Sawhney, Ph.D. - US grants

Assay of MCAK microtubule depolymerizing activity. During mitosis it is necessary that the duplicated chromosomes align on the mitotic spindle. Once all of the chromosomes are aligned at the metaphase plate, the sister anaphase chromatids split and migrate to their respective poles. The spindle is made up of microtubules, stiff polymers of a and B tubulin, which radiate from the centrosomes with a known polarity. Minus ends are bundled at the centrosome, whereas the plus ends are directed outward, some terminating at the centromeres of migrating mitotic chromosomes. Chromosomes can be moved with respect to the spindle by microtubule associated motor proteins. These molecules fall in two families, the Dyneins and Kinesins, which are known to have roles in vesicle transport, pigment movement, and transport of membrane-bound organelles. These two-headed motors hydrolyze ATP and utilize the energy to "walk" down microtubules, thus transporting their bound cargo. A given motor can walk eith er to the plus- or to the minus-end of microtubules. Three motor proteins have been isolated from centromeres: minus-end directed dynein, the plus-end directed kinesin CENP-E, and a poorly understood kinesin known as the Mitotic Centromere Associated Kinesin (MCAK.) MCAK was first described in 1995; however, its function still has not been determined unequivocally. It has not demonstrated an ability to actually move along microtubules but still appears to be important in chromosomal segregation. Preliminary studies suggest that the Xenopus homolog of MCAK, called XCCMI has a microtubule depolymerizing activity, possibly effecting chromosomal movement by modulating microtubule stability. We set out to assay MCAK for this activity. Stable fluorescent GMPCPP microtubules were polymerized and pipetted into a silanized flow cell. The flow cell was then washed with a lmg/ml solution of casein in order to remove unbound tubules and to coat glass surfaces. Finally, 0.51 M MCAK was flowed in (or heat-killed MCAK negative control.) Microtubules were then visualized by fluorescence microscopy, and a given set of microtubules was videotaped for three seconds every ten minutes, to enable assessment of length change. Experimental and control groups displayed microtubules shortening 0.93+0.09 microns/hr and 0.81+0.13 microns/hr respectively, not statistically significantly different. We are now assaying for ability to depolymerize free-floating tubules. Determining how such molecular motors work will also help understanding how cells achieve morphology, move, and exert forces on their environments.

High resolution 3D reconstruction of fibroblast traction in reconstituted collagen gels. Dense fibrous connective tissues, such as tendon, periosteum and the periodontal ligament, consist of two general components: extracellular matrix and cells. The matrix is composed largely of collagen, whose density, fibril diameter and orientation are the chief determinants of the tissue's mechanical properties. Fibers tend to be aligned parallel to the primary axis of tissue strain, which obviously improves resistance to tensile stresses. The etiology of this organization remains unknown. The cellular component of fibrous connective tissue is mainly fibroblasts which are responsible for both secreting and degrading collagen. Furthermore, fibroblasts have been shown to exert large forces on the matrix through which they crawl, far greater than that of faster moving cells such as leukocytes (Harris et al 1981) This excess force generated through the cytoskeleton brings about a phenomenon known as "traction." Gross examination in two dimensions, reveals that traction by tissue explant s can contract and align local collagen fibers, which may be important for connective tissue morphogenesis and maintenance of tissue integrity (Stopak and Harris, 1982). We have been imaging fibroblasts in collagen using differential interference contrast optics and confocal reflectance contrast microscopy in order to dissect the cellular structures and events involved in 3D fibroblast traction. Solubilized Type I collagen is "re-constituted" into collagen gels composed of a meshwork of collagen fibrils, ranging from 50-IOOnm in diameter, and detectable by reflectance (Friedl, 1997). The gels will be seeded with mouse dermal fibroblasts microinjected with rhodamine-labeled tubulin to a final concentration of 5nM. This low concentration (0.025% of the endogenous tubulin pool) yields "speckled" microtubules (Waterman-Storer and Salmon, 1998) which facilitate visualization of filament dynamics. Cells can also be pre-labeled with non-blocking fluorophore-linked anti-plintegrin antibody (Friedl, 1997). This setup will allow visualization of the three key elements involved in traction: the cytoskeleton, the extracellular matrix and the integrins that bridge the two. The challenges will be in: 1) simultaneously imaging these components, 2) resolving individual collagen fibrils and microtubule speckles in the crowded focal contact region, and 3) continuing visualization of individual living cells over sufficient time to observe tissue reorganization, without photobleaching or photodamaging cells.