Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes during Mitosis inDrosophila

Abstract

Cytoplasmic dynein is a multisubunit minus-end–directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immu-nofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

Functional Coordination of Three Mitotic Motors inDrosophilaEmbryos

“…Although we are aware of the caveats, as well as the strengths, of antibody microinjection experiments (Scholey, 1998), we consider it likely that the antibodies used in this study strongly and specifically inhibit the activities of dynein and KLP61F for the following reasons. First, the microinjection of our anti-DHC or anti-KLP61F antibodies produced effects on spindle pole positioning that are similar to those reported in KLP61F (Heck et al, 1993) and dynein (Robinson et al, 1999) mutants. Second, in the case of dynein, two different inhibitors, anti-DHC and p50 dynamitin, produced strikingly similar results.…”

“…In the first of these, it was shown that KLP61F and Ncd generate antagonistic forces to position mitotic spindle poles after NEB (Sharp et al, 1999b). More recently, it was shown that hypomorphic mutations in the dynein heavy chain reduced the extent of spindle pole separation during interphase-prophase in this system (Robinson et al, 1999). Although the roles of dynein, KLP61F, and Ncd are also reported here, the important novel feature of our work is to show how the activities of these motors are organized into an ordered pathway for spindle assembly and function.…”

“…These include the observations that the microinjection of antibodies inhibiting dynein function into mammalian cells gives rise to monoastral spindles containing side-by-side spindle poles (Vaisberg et al, 1993) and that dynein null mutants in budding yeast display defects in spindle elongation during anaphase B (Saunders et al, 1995). A very recent study has also shown that hypomorphic mutations of the dynein heavy chain in Drosophila inhibits spindle pole separation in early embryos (Robinson et al, 1999). Although it is plausible that dynein motors function during mitosis by driving MT-MT sliding as they do in the ciliary axoneme (Heald et al, 1996), there is also evidence that dynein becomes anchored on the cell cortex (Bloom et al, 1999) where it could slide astral MTs relative to the fixed cortex to separate the poles .…”

“…Two motors that might provide force to drive spindle pole separation during the early phases of mitosis are cytoplasmic dynein (Vaisberg et al, 1993;Robinson et al, 1999) and the bipolar kinesin KLP61F (Heck et al, 1993). Our previous studies suggest that KLP61F does not act until the later stages of mitosis (Sharp et al, 1999a,b), and this was supported by our current analyses, which indicate a rate for interphase-prophase spindle pole separation after the injec- tion of anti-KLP61F antibodies that is indistinguishable from controls (our unpublished results).…”

Interdependence of Filamentous Actin and Microtubules for Asymmetric Cell Division

“…Experiments with cytochalasin D indicated that actin caps are essential for maintaining an even nuclear spacing in the syncytium and for holding nuclei at the cortex (Sullivan et al, 1993;Foe et al, 2000). Recently, it was found that mutations in the dynein heavy chain gene disturb the regular spacing of the syncytial nuclei (Robinson et al, 1999). Nuclear positioning in the interphase syncytium therefore relies on a strict interdependence of microtubule and F-actin localization.…”