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First published online 14 September 2005
doi: 10.1242/dev.02039

Development 132, 4449-4459 (2005)
Published by The Company of Biologists 2005
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Cortical localization of the G{alpha} protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Katayoun Afshar1, Francis S. Willard2, Kelly Colombo1, David P. Siderovski2 and Pierre Gönczy1,*

1 Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL), CH-1066 Lausanne, Switzerland
2 Department of Pharmacology, Lineberger Comprehensive Cancer Center and Neuroscience Center, The University of North Carolina, Chapel Hill, NC 27599-7365, USA

* Author for correspondence (e-mail: pierre.gonczy{at}isrec.unil.ch)

Accepted 16 August 2005

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the G{alpha} proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gß protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two G{alpha} proteins can activate the same pathway and that their dual presence is normally needed to counter Gß{gamma}. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between G{alpha} proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.

Key words: C. elegans embryos, Spindle positioning, G protein


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