The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis

Cell Mol Life Sci. 2014 Feb;71(3):517-38. doi: 10.1007/s00018-013-1401-6. Epub 2013 Jun 27.

Abstract

Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They have been implicated, together with their interacting minus-end directed motor protein KIFC5A, in the regulation of centriole duplication in mammalian cells. Here we show that Nubp1 and Nubp2 are integral components of centrioles throughout the cell cycle, recruited independently of KIFC5A. We further demonstrate their localization at the basal body of the primary cilium in quiescent vertebrate cells or invertebrate sensory cilia, as well as in the motile cilia of mouse cells and in the flagella of Chlamydomonas. RNAi-mediated silencing of nubp-1 in C. elegans causes the formation of morphologically aberrant and additional cilia in sensory neurons. Correspondingly, downregulation of Nubp1 or Nubp2 in mouse quiescent NIH 3T3 cells markedly increases the number of ciliated cells, while knockdown of KIFC5A dramatically reduces ciliogenesis. Simultaneous double silencing of Nubp1 + KIFC5A restores the percentage of ciliated cells to control levels. We document the normal ciliary recruitment, during these silencing regimes, of basal body proteins critical for ciliogenesis, namely CP110, CEP290, cenexin, Chibby, AurA, Rab8, and BBS7. Interestingly, we uncover novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which we find enriched at the basal body and recruited independently of the Nubps or KIFC5A. Our combined results for Nubp1, Nubp2, and KIFC5A and their striking effects on cilium formation suggest a central regulatory role for these proteins, likely involving CCT/TRiC chaperone activity, in ciliogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Cycle / physiology*
  • Centrioles / metabolism*
  • Chlamydomonas
  • Chromatography, Liquid
  • Cilia / physiology*
  • Fluorescent Antibody Technique
  • GTP-Binding Proteins / metabolism*
  • Gene Knockdown Techniques
  • Immunohistochemistry
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Microscopy, Electron, Transmission
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Chaperones / metabolism
  • NIH 3T3 Cells
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tandem Mass Spectrometry

Substances

  • Intracellular Signaling Peptides and Proteins
  • KIFC5 protein, mouse
  • Microtubule-Associated Proteins
  • Molecular Chaperones
  • NUBP2 protein, mouse
  • Nubp1 protein, mouse
  • GTP-Binding Proteins