G Proteins and Spindle Positioning

Abstract

The mitotic spindle must be correctly positioned to allow symmetric or asymmetric cell division. In Caenorhabditis elegans in the first few cell divisions, asymmetric spindle positioning is due to the opposing forces generated as a consequence of the asymmetric distribution of PAR-2 (posterior) and PAR-3 (anterior). G proteins and LIN-5, a coiled-coil protein, contribute to the force generation and are downstream of the PARs, thus contributing to spindle positioning. Srinivasan et al. identified two G-protein regulator (GPR) proteins, GPR-1 and GPR-2, as part of the ~700-kD LIN-5 macromolecular complex. Coprecipitation and glutathione- S -transferase fusion protein pull-down experiments confirmed that the interaction between LIN-5 and GPR-1 and -2 was direct. RNA interference experiments showed that LIN-5 and GPR-1 and -2 deficiency produced similar spindle orientation defects. LIN-5 and GPR-1 and -2 colocalized at the cell cortex and the spindle asters, and this localization of GPR-1 and -2 required LIN-5. Genetic epistasis experiments indicated that LIN-5, GPR-1, and GPR-2 were essential for spindle movement, but not the establishment of polarity caused by PAR-2 and PAR-3. At later stages of development (4-cell stage), LIN-5 and GPR-1 and -2 accumulate asymmetrically at the cell cortex, this localization was lost in mutants for the genes mes-1 or src-1 , which encode tyrosine kinase-related proteins. Thus, both the PAR pathway, which establishes asymmetry in the first two divisions, and the mes-1 , src-1 pathway, which establishes asymmetry in response to external cues in later divisions, converge on LIN-5 and these GPRs. Because GPR-1 and -2 contain a GoLoco domain, which promotes interaction with Gα subunits of the G i/o type, the authors analyzed the polarity, spindle positioning, and mitotic phenotypes of mutants for the two Gα i/o genes ( goa-1 , gpa - 16 ) and the Gβ subunit ( gpb-1 ). Only Gα mutants mimicked the phenotypes observed in animals deficient in gpr-1 , gpr-2 or Lin-5 . Indeed, in vitro experiments suggested that GPR-1 and GPR-2 interacted with the guanosine-diphosphate (GDP)-bound form of GOA-1. The authors suggest that G proteins may be activated independently of a plasma membrane receptor (GPCR) and may control spindle positioning during cell division. D. G. Srinivasan, R. M. Fisk, H. Xu, S. van den Heuvel, A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C. elegans . Genes Dev . 17 , 1225-1239 (2003). [Abstract] [Full Text]