Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis

Cells. 2022 Jul 6;11(14):2127. doi: 10.3390/cells11142127.

Abstract

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Keywords: Asp; Dgt6; Drosophila; Eb1; Klp10A; Mars; Mast/Orbit/Chb; Mei-38; Patronin; S2 cells; colcemid; kinetochores; microtubule depolymerization; microtubule regrowth; mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Demecolcine / metabolism
  • Drosophila / metabolism
  • Drosophila Proteins* / genetics
  • Drosophila Proteins* / metabolism
  • Kinesins / genetics
  • Kinetochores* / metabolism
  • Mammals / metabolism
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Microtubules / metabolism
  • Mitosis
  • Spindle Apparatus / metabolism

Substances

  • Dgt6 protein, Drosophila
  • Drosophila Proteins
  • EB1 protein, Drosophila
  • Microtubule-Associated Proteins
  • patronin protein, Drosophila
  • KLP10A protein, Drosophila
  • Kinesins
  • Demecolcine

Grants and funding

This work was supported by grants from the Ministry of Education and Science of the Russian Federation (14.Z50.31.0005 to M.G.), from Russian Science Foundation (16-14-10288 to A.V.P.), from the Fundamental Scientific Research Program of the Ministry of Education and Science of the Russian Federation (project FWGZ-2021-0017 to A.V.P.), and from Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 20528 to M.G.; http://www.airc.it/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.