Optogenetic dissection of mitotic spindle positioning in vivo

Elife. 2018 Aug 15;7:e38198. doi: 10.7554/eLife.38198.

Abstract

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Keywords: C. elegans; LIN-5 (NuMA); cell biology; cell division; developmental biology; dynein; germline silencing; mitotic spindle positioning; optogenetics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Biomechanical Phenomena
  • Caenorhabditis elegans / embryology
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / radiation effects
  • Caenorhabditis elegans Proteins / metabolism
  • Cell Membrane / metabolism
  • Cell Membrane / radiation effects
  • Codon / genetics
  • Dyneins / metabolism
  • Embryo, Nonmammalian / metabolism
  • Embryo, Nonmammalian / radiation effects
  • GTP-Binding Protein alpha Subunits / metabolism
  • Gene Expression Regulation
  • Genes, Essential
  • Germ Cells / metabolism
  • Light
  • Optogenetics*
  • Spindle Apparatus / metabolism*
  • Transgenes

Substances

  • Caenorhabditis elegans Proteins
  • Codon
  • GTP-Binding Protein alpha Subunits
  • Dyneins