Single Particle Tracking of Surface Receptor Movement during Cell Division

We have used fluorescent latex beads to label membrane receptors on cultured NRK cells. Movement of individual beads during cell division was recorded with digital imaging techniques. Surface-bound beads showed no organized movement during metaphase but started to migrate toward the equator ,,ol min after anaphase onset, when chromosomes moved out of the equatorial region to create the interzone. The movement was most active in the central region of the cell near separating chromosomes, while beads located near the poles of the cell underwent primarily random motion. Most beads showed a surge in speed upon the passage of chromosomes, suggesting a possible link between chromosome separation and cortical reorganization. Furthermore, treatment of anaphase cells with cytochalasin D induced a rapid, simultaneous collapse of beads and cortical actin filaments into aggregates, indicating that the movement of beads was closely related to the reorganization of the actin cortex. In contrast to normal directional movement, cytochalasin-induced movement occurred in random directions and caused some beads in the equatorial region to move toward poles. Our results indicate that cytokinesis involves contractile activities, not only along the equator, but over a wide area of the actincontaining cortex. In addition, organized cortical activities appear to be temporally activated at anaphase onset, and spatially modulated by the spindle interzone or separating chromosomes. D SPITE many years of investigation, the mechanism of cytokinesis in animal cells remains poorly understood. It is generally recognized that the contractility of the cell cortex plays an important role (for reviews see Mabuchi, 1986; Rappaport, 1986; 1991; Salmon, 1989; Satterwhite and Pollard, 1992; Conrad and Schroeder, 1990; Fukui, 1993). Moreover, numerous ultrastructural, microinjection, and molecular biological studies have provided strong evidence for the involvement of actin and myosin II in the generation of cortical forces (Perry et al., 1971; Fujiwara and Pollard, 1976; Mabuchi and Okuno, 1977; Knecht and I.xmmis, 1987; DeLozaune and Spudich, 1987). Since both actin and myosin II become transiently organized in the equatorial region during cytokinesis, they may effect cell cleavage by generating a strong contractile force along the equatorial plane. What remains unclear is how the structure of actin and myosin becomes reorganized before and during cell division. Recent studies indicate that preexisting actin filaments can incorporate into the equatorial cortex of dividing cells (Cao and Wang, 1990a; Sanger et al., 1994), and that such incorporation involves the transport of cortical actin filaments toward the equator (Bray and White, 1988; Cao and Wang, Address all correspondence to Dr. Yu-li Wang, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545. Phone: (508) 845-2651; fax: (508) 842-3915. 1990b). Consistent with this process, a set of endogenous actin filaments have been found to align along the long axis of the cell in the region flanking the equator (Fukni and Inoue, 1991; Fishkind and Wang, 1993). These observations indicate that, to reach a better understanding of cytokinesis, it is important to address not only contractility along the cleavage furrow, but also a more global restructuring of the cell