Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis

• Published in: Cells (Basel, Switzerland) Vol. 11; no. 14; p. 2127
• 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
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 06.07.2022; MDPI
• Subjects: ASP protein; Centrosomes; Chromosomes; colcemid; Cold; Depolymerization; Drosophila; Genetic control; Insects; Kinetochores; Localization; Microsurgery; microtubule depolymerization; microtubule regrowth; Microtubules; Mitosis; Polymerization; Proteins; RNA-mediated interference; S2 cells; Spindles; Oregon; United States > US
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11142127

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.