During cell division, the mitotic spindle segregates chromosomes to the daughter cells and specifies the location of the cleavage plane. In a multicellular organism, developmental cues can alter the position of the mitotic spindle to generate cells of different sizes and to place cells into different spatial contexts. The Caenorhabditis elegans gene
lin-5 is essential for both chromosome segregation and correct spindle positioning during mitosis. In the early embryo,
lin-5 activity is required for posterior migration of the mitotic spindle and rotation of the centrosomes, activities needed to determine the correct division axis.
lin-5 encodes a novel coiled-coil protein which localizes to spindle microtubules, spindle asters and the cell cortex. To understand how developmental cues affect spindle position, we isolated LIN-5 protein complexes from C. elegans embryos. Gel filtration chromatography showed that LIN-5 protein is part of a complex of approximately 1 megadalton in size. Using LIN-5 specific monoclonal antibodies, we affinity purified endogenous LIN-5 from embryo lysates and analyzed these large-scale IPs by SDS/PAGE and silver staining. A specific 90 kDa band was identified conclusively as LIN-5 by tandem mass spectrometry. Peptides from a 60 kDa doublet were assigned to the product of either of two 98% identical genes located on chromosome III: C38C10.4 and F22B7.13 (here named
lap-1 and
lap-2 for LIN-5 associated proteins, also known as
ags-3.3 and
ags-3.2, respectively). RNAi analysis showed that
lap-1,2 are required for spindle migration and spindle rotations, similar to
lin-5. LAP-2 protein coelutes with LIN-5 in gel filtration chromotography and colocalizes with LIN-5 to spindle asters, kinetochore microtubules and the cell cortex. Interestingly, LAP-2 localization but not protein stability requires
lin-5 activity. The predicted LAP-1,2 proteins each contain a GPR/GoLoco motif. This motif is thought to bind the GDP-bound G i/o subunit of heterotrimeric G proteins and to affect the G guanine nucleotide exchange rates. Supporting the hypothesis that LAP-1,2 interact with G , combined RNAi of G subunits,
goa-1 and
gpa-16, yielded embryonic spindle positioning defects, as previously reported, which were strikingly similar to the
lin-5 or
lap-1,2 loss of function phenotype. GOA-1/G o and GPB-1/G proteins colocalize with LIN-5 and LAP-2 at the cell cortex. Additionally, LAP-2 binds GOA-1 in a GDP-dependent manner suggesting LAP-1,2 may regulate G activity. Together, these results suggest that the LAP-1,2 GoLoco proteins function with LIN-5 in a complex that regulates spindle-cortex interactions in a signaling pathway involving heterotrimeric G proteins.