Genetics and Cell Biology
The mechanisms of Drosophila spindle assembly: The role of kinetochore-driven microtubule formation
Gera Pavlova1,2,*, Julia Popova1,3,*, Alina Munzarova1,4,*, Julia Galimova1,*, Alena Razuvaeva1,4, Fioranna Renda5, Patrizia Somma5, Alexey Pindyurin1,4 and Maurizio Gatti5
1 Institute of Molecular and Cellular Biology, 8/2 Acad. Lavrentyev ave., Novosibirsk 630090, Russia
2 Kazan Federal University, Kazan, 420008, Russia
3 Institute of Cytology and Genetics, 10 Acad. Lavrentyev ave, Novosibirsk 630090, 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
The spindle is a complex and highly dynamic microtubule (MT)-based molecular machine that mediates precise chromosome segregation during both mitosis and meiosis. To form a spindle, centrosome-containing cells exploit 3 classes of microtubules: MTs nucleated by the centrosomes, MTs nucleated near the chromosomes/kinetochores and MT nucleated from preexisting MTs through the augmin-based pathway. Here we report our studies aimed at the identification and characterization of the genes/proteins required for kinetochore-driven MT growth. To identify these proteins we used Drosophila S2 cells and determined their proficiency in the process by analyzing spindle MT regrowth after cold- or colcemid-induced MT depolymerization.
We first asked whether the depolymerization conditions affect the pattern of MT regrowth. As expected, we found that after cold treatment MTs regrow much more rapidly than after colcemid treatment. In addition, we found that cold-induced MT disassembly at very low temperatures (-2°C) destroys kinetochore-driven but not centrosome-directed MT regrowth, while MT depolymerization at 0°C allows regrowth from both kinetochores and centrosomes. Colcemid-induced MT depolymerization strongly impaired centrosome-dependent MT nucleation but allowed MT regrowth from kinetochores. These results indicate that the kinetochore- and the centrosome-mediated MT assembly pathways exploit molecular mechanisms that are at least in part different.
We next focused on the cellular functions required for kinetochore-driven MT regrowth, and examined this process in prometaphases/metaphases from cell cultures depleted of specific spindle proteins by RNA interference (RNAi). These analyses identified several factors that positively affect the process (Eb1, Mast/Orbit, Mars, Mei-38 and Dgt6), as well as factors that appear to delay regrowth from kinetochores (Asp and Patronin). These results provide novel insight into the molecular mechanisms of kinetochore-driven MT growth and suggest a model for the regulation of the process. We are currently integrating these studies with in vivo observations of dividing cells expressing Cherry-tagged tubulin or other fluorescently labeled proteins involved in MT regrowth from kinetochores. We are also using transmission electron microscopy to characterize normal mitosis and MT regrowth in S2 cells.