Functional Coordination of Three Mitotic Motors inDrosophilaEmbryos

Abstract

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear.Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is downregulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B. INTRODUCTIONThe segregation of chromosomes during mitosis depends on the action of a self-organizing, bipolar machine called the mitotic spindle. It is now established that the formation and function of the mitotic spindle requires numerous microtubule (MT)-based motor proteins (Hoyt and Geiser, 1996;Vale and Fletterick, 1997). Although the identities of many of these mitotic motors are becoming clear, their specific functional interrelationships have been extremely difficult to ascertain.Among all mitotic movements, the positioning of spindle poles during the assembly and elongation of the bipolar mitotic spindle may require the greatest degree of cooperation between different motors. This process is particularly complex because it occurs in a pathway consisting of several, temporally distinct stages, during which the organization of spindle microtubules and the general environment of the cell change dramatically (McIntosh and McDonald, 1989). The members of at least three families of MT motors are thought to play important roles in this pathway. These are the bipolar kinesins, the C-terminal kinesins, and cytoplasmic dynein.The bipolar (or BimC) kinesins (Vale and Fletterick, 1997) comprise a family of plus-end-directed motors, which have a bipolar morphology with motor domains at both ends of a central rod (Cole et al., 1994; Kashina et al., 1996a,b; Gordon and Roof, 2000). Functionally, these motors are thought to play a role in either the assembly or maintenance of spindle bipolarity, because their inhibition results in the formation of monopolar mitotic spindles (Enos and Morris, 1990;Hagan and Yanagida, 1990;Roof et al., 1991;Hoyt et al., 1992;Sawin et al., 1992;Heck et al., 1993;Blangy et al., 1995;Sharp et al., 1999b). Support for a role for bipolar kinesins in spindle maintenance but not assembly comes from the recent findings that inhibiting the Drosophila bipolar kinesin KLP61F does not pr...

Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization inDrosophila

“…Mouse-anti-P10 (Dick et al, 1996) at 1/500 and rat-anti-DDLC1 at 1/200 (Ghosh-Roy et al, 2004) were used for the immunolocalization of DDLC1. The monoclonal anti-DHC (Sharp et al, 2000) and polyclonal anti-Shibire (Estes et al, 1996) were used at 1/10 and 1/200 dilutions, respectively. …”

The perpetual movements of anaphase

“…On the other hand, injection of dynein antibodies or overexpression of human p50 dynamitin in Drosophila syncytial embryos was reported to affect chromosome movement by impairing both anaphase A and B [96,97]. However, from these studies and due to the rapid syncytial divisions, it was not clear whether the observed effects on anaphase A were truly due to a requirement for kinetochore dynein in chromosome movement or to an indirect effect caused by "forced" mitotic exit.…”

“…separation of spindle poles by cortical mediated pulling forces on astral microtubules), also known as anaphase C [212]. The microtubule sliding mechanism proposed by McIntosh and colleagues (1969) explains at least part of the spindle elongation in many systems, including diatoms, yeasts and Drosophila embryos [96,[213][214][215][216]. At the molecular level, the players involved in each system might be different, although their modus operandi must be essentially the same: cross-linking, sliding and eventually growth/stabilization of interpolar microtubules of opposite polarity.…”

“…At the molecular level, the players involved in each system might be different, although their modus operandi must be essentially the same: cross-linking, sliding and eventually growth/stabilization of interpolar microtubules of opposite polarity. These functions are provided by MAPs that localize to the spindle midzone, such as members of the PRC1, XMAP215 and CLASP families, as well as by force-producing motors of the kinesin-5 or -6 families [96,[217][218][219][220][221][222][223][224][225].…”

“…These data indicated that in some systems, pulling forces acting on astral microtubules are the main drivers of spindle elongation, whereas spindle midzone microtubules resist or govern the rate of pole separation. Cytoplasmic dynein located at the cell cortex has been implicated in this astral MT pulling mechanism [89,90,96,[233][234][235]. On the other hand, proteins required for microtubule polymerization and growth/stabilization of interpolar microtubules [236], such as CLASPs [219], coordinated with the activity of kinesin-5 at the spindle midzone, might work as governors [237].…”