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:
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. We thank Maria B. Schwartz (Berkaeva) for her help during the early stages of this work. We also thank Mikhail O. Lebedev and Tatiana D. Dubatolova for their technical assistance. DNA sequencing and confocal microscopy analysis were performed using resources provided by the Molecular and Cellular Biology core facility of the Institute of Molecular and Cellular Biology SB RAS.
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 - Dgt6
KW - Drosophila
KW - Eb1
KW - Klp10A
KW - Mars
KW - Mast/Orbit/Chb
KW - Mei-38
KW - Patronin
KW - S2 cells
KW - colcemid
KW - kinetochores
KW - microtubule depolymerization
KW - microtubule regrowth
KW - mitosis
UR - http://www.scopus.com/inward/record.url?scp=85133410164&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85133410164&partnerID=8YFLogxK
U2 - 10.3390/cells11142127
DO - 10.3390/cells11142127
M3 - Article
AN - SCOPUS:85133410164
SN - 2073-4409
VL - 11
JO - Cells
JF - Cells
IS - 14
M1 - 2127
ER -