title: Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division.
abstract: Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha 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 Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. 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 Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.
authors.affiliation: Swiss Institute for Experimental Cancer Research (ISREC Institute of Technology (EPFL), CH-1066 Lausanne, Switzerland.
authors.forename: Katayoun
authors.initials: K
authors.lastname: Afshar
authors.forename: Francis S
authors.initials: FS
authors.lastname: Willard
authors.forename: Kelly
authors.initials: K
authors.lastname: Colombo
authors.forename: David P
authors.initials: DP
authors.lastname: Siderovski
authors.forename: Pierre
authors.initials: P
authors.lastname: Gönczy
mesh_terms: D000818:Animals; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013997:Time Factors
chemical_list: D029742:Caenorhabditis elegans Proteins; C475458:G protein regulator 1, C elegans; C475459:G protein regulator 2, C elegans; D044385:GTP-Binding Protein alpha Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C413418:RIC-8 protein, C elegans; C480838:GPA-16 protein, C elegans