The pattern of cell division is important for development, both for partitioning cytoplasmic factors during asymmetric divisions and for setting up cell contacts required for cell signaling. In C. elegans embryos, the first division produces a larger anterior AB cell and a smaller posterior P1 cell. The orientation of this asymmetric division is due to rotation of the nuclear-centrosome complex onto the polarized axis; the spindle then moves towards the posterior during metaphase/anaphase. Similar movements occur during the asymmetric division of P1. Spindle positioning is dependent on astral microtubules and their interactions with the cortex, as well as PAR proteins that are asymmetrically localized at the cortex.
We previously identified a temperature sensitive allele of the
spn-2 gene in screens for maternal effect mutations that result in spindle positioning defects. In embryos from
spn-2 mutant mothers raised at 20°C, defects in AB and P1 spindle orientation were seen. Stronger defects were observed in embryos analyzed at 25°C. Some one-cell embryos exhibited a failure of nuclear rotation, while in others rotation occurred but then the spindle showed excessive posterior movement during anaphase. Additionally, some two-cell
spn-2 embryos showed mispositioned nuclei, ectopic furrows, or centrosomes detached from the nucleus. Together these phenotypes lead to the hypothesis that SPN-2 is required for multiple microtubule-dependent processes.
To understand its role at the molecular level, we cloned the
spn-2 gene. A nonsense mutation was found in the F56F3.1 gene in
spn-2 worms and F56F3.1 RNAi embryos phenocopy
spn-2 mutants. In addition, transformation with the F56F3 cosmid rescued
spn-2 mutants. The F56F3.1 protein shows weak homology to a translation initiation factor 4E binding protein. To gain more insight into the role of
spn-2 in spindle positioning and whether SPN-2 acts via translation, we are quantifying the effects of loss of SPN-2 function on microtubule behavior and examining the localization and amounts of microtubule and centrosome associated proteins. We are also raising antibodies to determine the cellular localization of SPN-2.