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A new method for accurate measurement of poleward microtubule flux in metaphase spindles of Drosophila S2 cells

Name
Alina
Surname
Munzarova
Scientific organization
Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia
Academic degree
no
Position
PhD student
Scientific discipline
Life Sciences & Medicine
Topic
A new method for accurate measurement of poleward microtubule flux in metaphase spindles of Drosophila S2 cells
Abstract
The microtubule (MT) flux is the poleward movement of spindle MTs caused by their plus end polymerization coupled with minus end depolymerization. The flux regulates both spindle assembly and anaphase chromosome movement. We developed the “double spindle photobleaching (DSP)” method for measuring the flux in Drosophila S2 cell spindles containing Cherry-tagged MTs. This method allows a reliable measure of the flux rate over a 2-3-fold longer time than the standard photobleaching-based methods.
Keywords
mitotic spindle, microtubule flux, FRAP, Drosophila
Summary

A new method for accurate measurement of poleward microtubule flux in metaphase spindles of Drosophila S2 cells

 

Alina Munzarova1,2, Julia Popova1,3, Alena Razuvaeva1,2, Victor Shloma1, Maurizio Gatti1,4 and Leonid Omelyanchuk1,2

 

1 Institute of Molecular and Cellular Biology, 8/2 Acad. Lavrentyev ave., Novosibirsk 630090, Russia

2 Novosibirsk State University, 2 Pirogov str., Novosibirsk 630090, Russia

3 Institute of Cytology and Genetics, 10 Acad. Lavrentyev ave, Novosibirsk 630090, Russia

4 Department of Biology and Biotechnology, Sapienza, University of Rome, 00185 Rome, Italy

 

The spindle is a highly dynamic microtubule (MT)-based machine that mediates chromosome segregation during mitotic and meiotic cell division. One of the dynamic parameters that characterize the spindle is the poleward MT flux, namely the continuous translocation of MTs toward the spindle poles caused by MT polymerization at plus ends coupled with depolymerization at minus ends. Poleward MT flux is observed in all higher eukaryotes and contributes to the regulation of spindle length and anaphase chromosome movement. One of the standard methods to assess the flux rate consists in generating a photobleached area across a spindle that contains fluorescently-tagged MTs, and then measuring the velocity of the poleward movement of the non-fluorescent stripe. However, this method only permits rapid measurements of the flux, because the fluorescence of the bleached stripe recovers rapidly due to the spindle MT turnover. Here we describe a modification of the current “single stripe photobleaching (SSP)” method for flux measurement. We photobleached two large areas at the opposite sides of the metaphase plate in Drosophila S2 cells expressing Cherry-tagged tubulin, leaving unbleached only the area near the chromosomes. We then measured the speed with which the fluorescent MTs move towards the poles. We found that this “double spindle photobleaching (DSP)” method allows a measure of the flux over a 2-3-fold longer time than the SSP method, providing a reliable evaluation of the flux rate.