"Developmental control of cleavage plane specification"
Cell division during cytokinesis occurs in a plane perpendicular to and generally midway through the mitotic spindle. Thus, the position of the mitotic spindle affects both the polarity and directionality of cell division, aspects of cell division that are critical in the generation of daughter cells that differ in developmental potential. We study the lin-5 gene in C. elegans to further elucidate the roles of the spindle apparatus in chromosome segregation and cleavage plane specification.
Our previous results have shown that lin-5 is required for chromosome movements, spindle positioning and correct alternation of the S and M phases of the cell cycle. lin-5 encodes a coiled-coiled protein that localizes to spindle asters, kinetochore microtubules and the cell periphery.
LIN-5 (green) localization in an early embryo. DNA is in red.
To further reveal the molecular function of LIN-5, we have focused on identifying its binding partners. Based on gel filtration chromatography, LIN-5 is part of a large molecular weight protein complex. Immunoprecipitations with anti-LIN-5 monoclonal antibodies followed by SDS-PAGE and silver staining revealed at least eight other co-precipitated proteins. We identified two of these LIN-5 associated proteins by tandem mass spectrometry as the product(s) of two paralogous genes, gpr-1 and gpr-2.
Inactivation of either gene by RNA interference caused mitotic defects that were strikingly similar to the effects of lin-5 loss of function. Furthermore, GPR-1,2 colocalized with LIN-5 to spindle asters, kinetochore microtubules and the cell cortex in a lin-5-dependent manner. GPR-1 and GPR-2 each contain a GoLoco/G Protein Regulatory (GPR) motif that has been shown to interact with the Gαi/o subunit of heterotrimeric G proteins. A number of observations support that GPR-1,2 interacts with C. elegans Gα proteins. Combined RNAi of two Gα subunits, goa-1 and gpa-16, yielded embryonic spindle positioning defects, as previously reported, which were strikingly similar to the lin-5 and gpr-1,2 loss of function phenotype. GOA-1 Gα colocalized with LIN-5 and GPR-1,2 at the cell cortex, and GPR-1 bound GOA-1 in a GDP-dependent manner. These results support a model in which interactions between LIN-5, GPR-1,2 and GOA-1 Gα are required for generating the spindle forces needed for chromosome segregation (Srinivasan et al. G&D 2003).
Developmental cues polarize the cytoskeleton, which likely affects LIN-5/GPR/Gα function and creates asymmetric pulling forces on the spindle asters. These forces determine the position of the mitotic spindle, and thereby the cleavage plane of the cell.
We have initiated a novel project to characterize genetic control of cell polarity establishment in conjunction with an asymmetric division pattern. This study focuses on the stem-cell like division pattern of the "seam" cells in de larval epidermis. Among the attractive aspects of this lineage are the roles for cell-cell communication and apparent contribution of Wnt/Wg pathway components and homologs of the DLG and Scribble tumor suppressor proteins.
We also continue our studies of asymmetric division in the early embryo. This project incorporates extensive use of mass spectrometry, large-scale RNAi and yeast two-hybrid approaches. We have also initiated in vitro experiments with baculovirus expressed proteins purified from insect cells. Combined with in vivo observations of the role of each component in C. elegans embryos, these in vitro studies will allow us to further establish the molecular interactions and regulatory steps involved in developmentally regulated asymmetric division. In collaboration with the Theoretical Biology department we aim to mathematically model aspects of these divisions.