Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division
| Autor: | Afshar, K., Willard, F. S., Colombo, K., Siderovski, D. P., Gönczy, P. |
|---|---|
| Předmět: |
Guanine Nucleotide Exchange Factors/metabolism
Time Factors Blastomeres/cytology/metabolism Caenorhabditis elegans Proteins/genetics/*metabolism Caenorhabditis elegans/*cytology/embryology/genetics/*metabolism Extramural macromolecular substances Guanine Nucleotide Dissociation Inhibitors/metabolism Research Support P.H.S Cerebral Cortex/cytology/embryology/metabolism N.I.H Nonmammalian/cytology/metabolism Nuclear Proteins/genetics/*metabolism stomatognathic system Gene Expression Regulation Embryo GTP-Binding Protein alpha Subunits/genetics/*metabolism Animals Developmental U.S. Gov't Non-U.S. Gov't Cell Division Protein Binding |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|