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

Name
Gera
Surname
Pavlova
Scientific organization
Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia
Academic degree
-
Position
PhD student
Scientific discipline
Life Sciences & Medicine
Topic
Genetic control of kinetochore-driven microtubule growth: An RNAi-based analysis in Drosophila S2 cells
Abstract
To understand the mechanisms of chromosome/kinetochore-driven microtubule (MT) formation we analyzed MT regrowth after MT depolymerization in Drosophila S2 cells. MT regrowth assays were performed in cells subjected to RNAi against 10 individual spindle components. We identified factors that promote MT regrowth and factors that inhibit this process. Our results provide a sound starting point to develop a molecular model for the regulation of kinetochore-driven MT growth.
Keywords
Drosophila S2 cells, mitosis, spindle, kinetochores, microtubule regrowth assay
Summary

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

 

Julia Popova1,2,*, Gera Pavlova1,3,*, Alina Munzarova1,4,*, Fioranna Renda5, Patrizia Somma5, Alexey Pindyurin1,4 and Maurizio Gatti1,5

 

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.