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In vitro reconstitution of branching microtubule nucleation.

Elife. 2020 Jan 14;9
Ammarah Tariq 1 , Lucy Green 1 , J Charles G Jeynes 1 , Christian Soeller 1 , James G Wakefield 1
Ammarah Tariq 1 , Lucy Green 1 , J Charles G Jeynes 1 , Christian Soeller 1 , James G Wakefield 1

[No authors listed]

Author information
  • 1 Living Systems Institute, University of Exeter, Exeter, United Kingdom.

摘要


Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed 'branching' MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

KEYWORDS: D. melanogaster, cell biology, microtubule, mitosis, mitotic spindle