[
European Worm Meeting,
2004]
Generation of asymmetry in the 1-cell embryo of C. elegans establishes the anterio- posterior axis and is necessary for the position and orientation of the mitotic spindle. The sperm-contributed centrosomes define the posterior of the embryo probably via interaction of the microtubules (MTs) with the actin rich cortex. This leads to the asymmetric cortical localisation of conserved polarity proteins, such as the PAR proteins, to anterior or posterior cortical domains. These proteins in turn control the asymmetric positioning of the first spindle through regulation of the microtubule function. To identify new molecules required for these early events, we carried out a genome-wide RNAi screen to find embryonic lethal genes. We then videorecorded the first three cell divisions after RNAi of these genes and examined early embryonic events to identify genes specifically required for early cell polarity. F22D6.6 was identified in the screen as a gene whose inactivation results in a strong posterior spindle displacement defect. It codes for a protein of unknown function containing two Tudor domains, which are thought to mediate protein-protein interaction, but no other domains that suggest a biochemical activity. This domain is found in a set of different genes, from the Drosophila founding member Tudor, involved in germ line formation to the product of the spinal muscular-atrophy determining Survival Motor Neuron (SMN) gene. In F22D6.6(RNAi) embryos, pronuclear migration is normal, but the pronuclear/centrosome complex fails to undergo complete rotation. The spindle aligns late along the A/P axis and is pulled rapidly too far to the posterior. PAR polarity is also disturbed as PAR-2 often localises to both sides of the embryo and shows a strong association with the centrosome instead of being posteriorly restricted. In addition, F22D6.6(RNAi) induces cell cycle and germline defects. We will present our progress in understanding F22D6.6 function.
[
International Worm Meeting,
2003]
Generation of asymmetry in the 1-cell embryo of C. elegans establishes the anterio- posterior axis and is necessary for the position and orientation of the mitotic spindle. Microtubules (MTs) play a central role in these processes. For example, MTs emanating from the sperm-contributed centrosomes define the posterior of the embryo through interaction with the actin rich cortex, leading to the asymmetric cortical localisation of a set of conserved polarity proteins, the PAR proteins, to anterior or posterior cortical domains. Asymmetric spindle positioning also involves MT regulation. To identify new molecules required for these MT functions, we carried out a genome-wide RNAi screen to find embryonic lethal genes. We then videorecorded the first three cell divisions after RNAi of these genes and examined early embryonic events to identify genes specifically required for early cell polarity. F22D6.6 was identified in the screen as a gene whose inactivation results in a strong posterior spindle displacement defect. It codes for a protein of unknown function containing two Tudor domains, which are thought to mediate protein-protein interaction, but no other domains that suggest a biochemical activity. In F22D6.6(RNAi) embryos, pronuclear migration is normal, but the pronuclear/centrosome complex fails to undergo complete rotation. The spindle aligns late along the A/P axis and is pulled rapidly too far to the posterior. Other aspects of polarity appear to be disturbed too, as the posterior polarity cue PAR-2 often localises to both sides of the embryo and shows a strong association with the centrosome. We are examining the localisation of the LET-99 protein, which is required for rotation, in F22D6.6(RNAi) embryos. The localisation of the F22D6.6 protein is currently under study via antibody staining and a GFP construct. To identify interacting partners we are carrying out a two-hybrid screen. The molecular nature of these interactors will help to understand F22D6.6 function and their role will be analysed using RNAi, DIC video-recording and assaying the localisation of polarity markers.