Evidence for dynein and astral microtubule-mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

Mol Biol Cell. 2013 Apr;24(7):901-13. doi: 10.1091/mbc.E12-06-0458. Epub 2013 Feb 6.

Abstract

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule-mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

Publication types

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

MeSH terms

  • Animals
  • Antigens, Nuclear / genetics
  • Antigens, Nuclear / metabolism*
  • Biological Transport
  • Blotting, Western
  • COS Cells
  • Cell Cycle Proteins
  • Cell Line
  • Cell Line, Tumor
  • Cytoplasmic Dyneins / genetics
  • Cytoplasmic Dyneins / metabolism*
  • Fluorescence Recovery After Photobleaching
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Microscopy, Fluorescence
  • Microtubules / drug effects
  • Microtubules / metabolism
  • Mitosis*
  • Multiprotein Complexes / metabolism
  • Mutation
  • Nocodazole / pharmacology
  • Nuclear Matrix-Associated Proteins / genetics
  • Nuclear Matrix-Associated Proteins / metabolism*
  • Protein Binding / drug effects
  • RNA Interference
  • Spindle Apparatus / metabolism*
  • Tubulin Modulators / pharmacology

Substances

  • Antigens, Nuclear
  • Cell Cycle Proteins
  • DYNC1H1 protein, human
  • GPSM2 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Multiprotein Complexes
  • NUMA1 protein, human
  • Nuclear Matrix-Associated Proteins
  • Tubulin Modulators
  • Cytoplasmic Dyneins
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Nocodazole