Genetic control of kinetochore-driven microtubule growth: An RNAi-based analysis in Drosophila S2 cells

Genetic control of kinetochore-driven microtubule growth: An RNAi-based analysis in Drosophila S2 cells

Julia Popova^1,2,*, Gera Pavlova^1,3,*, Alina Munzarova^1,4,*, Fioranna Renda⁵, Patrizia Somma⁵, Alexey Pindyurin^1,4 and Maurizio Gatti^1,5

¹ Institute of Molecular and Cellular Biology, 8/2 Acad. Lavrentyev ave., Novosibirsk 630090, Russia

² Institute of Cytology and Genetics, 10 Acad. Lavrentyev ave, Novosibirsk 630090, Russia

³ Kazan Federal University, Kazan, 420008, Russia

⁴ Novosibirsk State University, 2 Pirogov str., Novosibirsk 630090, Russia

⁵ Department of Biology and Biotechnology, Sapienza, University of Rome, 00185 Rome, Italy

^* equal contribution

Chromosome/kinetochore-driven microtubule formation is essential for proper spindle assembly. To dissect the mechanisms underlying this process we analyzed spindle microtubule (MT) regrowth after cold- or colcemid-induced MT depolymerization in Drosophila S2 cells depleted of individual spindle components. Specifically, we used RNAi to deplete 3 MT-destabilizing kinesins (Klp10A, Klp59C and Klp67A), 2 proteins that favor MT plus end growth (Eb1, Mast/Orbit/Clasp), 2 proteins that bind and stabilize the kinetochore fibers (Mars/HURP and Mei-38/TPX2), 2 proteins that specifically associate with the MT minus ends (Asp and Patronin), and Dgt6, a component of the augmin complex that mediates lateral MT growth from preexisting MTs. MT regrowth assays performed in prometaphase/metaphase cells showed that depletion of the MT-destabilizing kinesins does not affect kinetochore-driven MT growth, which is instead reduced by the loss of Eb1, Mast/Orbit, Mars, Mei-38 or Dgt6. Surprisingly, we found that depletion of either Asp or Patronin increases the rate of MT regrowth from the kinetochores. The current model on kinetochore-driven MT growth in cells not exposed to MT depolymerization suggests that kinetochores capture the plus ends of the MTs that form in their vicinity, and that polymerization of these plus ends leads to formation of kinetochore fibers with the minus ends pointing away from the chromosomes. Our results are consistent with this model and identify several factors that are necessary for growth and stabilization of the MT bundles emanating from the kinetochores. However, the precise roles of Asp and Patronin in the regulation of kinetochore-driven MT regrowth remain to be defined.