Spindle Pole Organization inDrosophilaS2 Cells by Dynein,Abnormal SpindleProtein (Asp), and KLP10A

Abstract

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. We evaluated the functions of the dynein-dynactin complex by using RNA interference (RNAi)-mediated depletion of distinct subunits in Drosophila S2 cells. We observed a striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindle protein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein-dynactin, we observed subtle but significant mislocalization of KLP10A and Asp, suggesting that dynein-dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopus extracts to Drosophila cultured cells and suggest that common pathways contribute to spindle pole organization and length determination.

Kinesin-5–dependent Poleward Flux and Spindle Length Control inDrosophilaEmbryo Mitosis

“…Although we cannot rule out an influence of technical differences between the two studies, we suspect that they reflect genuine system-specific differences, akin to the different consequences of loss of Ncd function in embryos versus cultured cells of Drosophila (Sharp et al, 2000a;Goshima et al, 2005a;Morales-Mulia and Scholey, 2005).…”

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

“…Loss of a kinesin-14 function may cause different mitotic and meiotic spindle defects even within the same organism. For example, in Drosophila S2 cells and female meiosis, loss of Ncd by RNA interference (RNAi) or null mutants causes disorganization of spindle poles (Theurkauf and Hawley, 1992;Morales-Mulia and Scholey, 2005), whereas in embryos, it causes changes in spindle length and a decrease in the persistence of steady-state structures (Sharp et al, 1999;Brust-Mascher and Scholey, 2002).…”

“…Loss of a kinesin-14 function may cause different mitotic and meiotic spindle defects even within the same organism. For example, in Drosophila S2 cells and female meiosis, loss of Ncd by RNA interference (RNAi) or null mutants causes disorganization of spindle poles (Theurkauf and Hawley, 1992;Morales-Mulia and Scholey, 2005), whereas in embryos, it causes changes in spindle length and a decrease in the persistence of steady-state structures (Sharp et al, 1999;Brust-Mascher and Scholey, 2002).Evidence suggests that kinesins-14 act by driving the sliding of parallel or antiparallel MT bundles in different areas of the spindle (for review, see Sharp et al, 2000b,c;McIntosh et al, 2002;Gadde and Heald, 2004). These motors have two MT binding sites, an ATP-dependent site in the motor domain and an ATP-independent site in the tail, and they have been shown to induce MT bundling (McDonald et al, 1990;Chandra et al, 1993;Karabay and Walker, 1999;Matuliene et al, 1999).…”

“…These motors have two MT binding sites, an ATP-dependent site in the motor domain and an ATP-independent site in the tail, and they have been shown to induce MT bundling (McDonald et al, 1990;Chandra et al, 1993;Karabay and Walker, 1999;Matuliene et al, 1999). Recent small interfering RNA (siRNA) studies demonstrate that knockdown of the kinesin-14 family member Ncd results in the formation of splayed mitotic spindle poles, suggesting that the activity of this motor is required for focusing of the MTs at the pole (Goshima and Vale, 2003;Morales-Mulia and Scholey, 2005). It has been found that loss of kinesin-14 function in S. cerevisiae and Drosophila leads to an increase in the spindle length and that it rescues spindle pole separation defects seen in cells lacking members of the kinesin-5 family (Saunders and Hoyt, 1992;Saunders et al, 1997;Sharp et al, 1999;Sharp et al, 2000a) and that these two motors oppose each other in in vitro motility assays (Tao et al, 2006), suggesting that the balance of their activities is required for maintaining the correct distance between the spindle poles.…”

The perpetual movements of anaphase

“…Indeed, it has been estimated that every vertebrate kinetochore is tangentially associated with 3-8 non-kinetochore microtubules [200]. Additionally, several proteins such as dynein and its co-factor NuMA, kinesin-14 members or the Drosophila microtubule associated protein ASP have also been implicated in spindle pole focusing by mediating microtubule crosslinking [91,201,202]. On this regard, we favor a general distribution of microtubule cross-linkers along the length of the k-fiber, as this would explain why chromosomes lie at the equator after U.V or laser microbeam irradiation of the respective k-fibers during metaphase [52,203].…”