The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein

Dev Biol. 2016 Apr 15;412(2):288-297. doi: 10.1016/j.ydbio.2016.02.020. Epub 2016 Feb 26.

Abstract

PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization.

Keywords: Asymmetric division; C. elegans; Embryo; LET-99; PAR-5.

Publication types

  • Research Support, American Recovery and Reinvestment Act
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • Embryo, Nonmammalian / embryology
  • Embryo, Nonmammalian / metabolism*
  • Microscopy, Confocal
  • Mutation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • RNA Interference
  • Time-Lapse Imaging
  • Two-Hybrid System Techniques

Substances

  • Caenorhabditis elegans Proteins
  • LET-99 protein, C elegans
  • par-5 protein, C elegans
  • PAR-1 protein, C elegans
  • Protein-Serine-Threonine Kinases