The Molecular Architecture of the Yeast Spindle Pole Body Core Determined by Bayesian Integrative Modeling

Mol Biol Cell. 2017 Nov 7;28(23):3298-3314. doi: 10.1091/mbc.E17-06-0397. Epub 2017 Aug 16.

Abstract

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

MeSH terms

  • Bayes Theorem
  • Cell Cycle
  • Centrosome / metabolism
  • Computer Simulation
  • Crystallography, X-Ray / methods
  • Microtubule-Organizing Center / metabolism
  • Microtubules / metabolism
  • Mitosis
  • Nuclear Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Spindle Apparatus / metabolism
  • Spindle Pole Bodies / metabolism*
  • Spindle Pole Bodies / physiology*
  • Structure-Activity Relationship
  • X-Ray Diffraction / methods

Substances

  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins