TY - JOUR

T1 - Genetic Control of Kinetochore-Driven Microtubule Growth in Drosophila Mitosis

AU - Popova, Julia V.

AU - Pavlova, Gera A.

AU - Razuvaeva, Alyona V.

AU - Yarinich, Lyubov A.

AU - Andreyeva, Evgeniya N.

AU - Anders, Alina F.

AU - Galimova, Yuliya A.

AU - Renda, Fioranna

AU - Somma, Maria Patrizia

AU - Pindyurin, Alexey V.

AU - Gatti, Maurizio

N1 - Funding Information: 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. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2022/7/1

Y1 - 2022/7/1

N2 - 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.

AB - 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.

KW - Asp

KW - colcemid

KW - Dgt6

KW - Drosophila

KW - Eb1

KW - kinetochores

KW - Klp10A

KW - Mars

KW - Mast/Orbit/Chb

KW - Mei-38

KW - microtubule depolymerization

KW - microtubule regrowth

KW - mitosis

KW - Patronin

KW - S2 cells

KW - Drosophila Proteins/genetics

KW - Mitosis

KW - Spindle Apparatus/metabolism

KW - Demecolcine/metabolism

KW - Kinesins/genetics

KW - Microtubule-Associated Proteins/genetics

KW - Drosophila/metabolism

KW - Kinetochores/metabolism

KW - Microtubules/metabolism

KW - Animals

KW - Mammals/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85133410164&partnerID=8YFLogxK

U2 - 10.3390/cells11142127

DO - 10.3390/cells11142127

M3 - Article

C2 - 35883570

AN - SCOPUS:85133410164

VL - 11

JO - Cells

JF - Cells

SN - 2073-4409

IS - 14

M1 - 2127

ER -