Genetic Control of Kinetochore-Driven Microtubule Growth in IDrosophila/I Mitosis

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...

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Published in:Cells (Basel, Switzerland) Switzerland), 2022-07, Vol.11 (14)
Main Authors: Popova, Julia V, Pavlova, Gera A, Razuvaeva, Alyona V, Yarinich, Lyubov A, Andreyeva, Evgeniya N, Anders, Alina F, Galimova, Yuliya A, Renda, Fioranna, Somma, Maria Patrizia, Pindyurin, Alexey V, Gatti, Maurizio
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Analysis
Control
Drosophila
Genetic aspects
Identification and classification
Kinetochores
Methods
Microtubules
Polymerization
Structure
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container_issue 14
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container_title Cells (Basel, Switzerland)
container_volume 11
creator Popova, Julia V
Pavlova, Gera A
Razuvaeva, Alyona V
Yarinich, Lyubov A
Andreyeva, Evgeniya N
Anders, Alina F
Galimova, Yuliya A
Renda, Fioranna
Somma, Maria Patrizia
Pindyurin, Alexey V
Gatti, Maurizio
description 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.
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subjects Analysis
Control
Drosophila
Genetic aspects
Identification and classification
Kinetochores
Methods
Microtubules
Polymerization
Structure
title Genetic Control of Kinetochore-Driven Microtubule Growth in IDrosophila/I Mitosis
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