Accurate measurement of poleward microtubule flux in the spindle of Drosophila S2 cells

Cell Biol Int. 2016 Sep;40(9):984-90. doi: 10.1002/cbin.10638. Epub 2016 Jul 12.

Abstract

The spindle microtubule (MT) flux is the continuous translocation of MTs toward the spindle poles caused by MT polymerization at plus ends coupled to depolymerization at minus ends. Poleward flux is observed in both mitotic and meiotic spindles; it is evolutionarily conserved and contributes to the regulation of spindle length and anaphase chromosome movement. MT photobleaching is a tool frequently used to measure poleward flux. Spindles containing fluorescently tagged tubulin are photobleached to generate a non-fluorescent stripe, which moves toward the spindle poles allowing a measure of the flux. 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 photobleaching-based method for flux measurement. We photobleached two large areas at the opposite sides of the metaphase plate in spindles of 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 toward the poles. We found that this method allows a measure of the flux over a two- to threefold longer time than the "single stripe" method, providing a reliable evaluation of the flux rate.

Keywords: DSP method; Drosophila cells; metaphase; microtubule flux; mitosis.

MeSH terms

  • Anaphase / physiology
  • Animals
  • Chromosome Segregation
  • Drosophila
  • Kinesin / metabolism
  • Kinetochores / metabolism
  • Metabolic Flux Analysis / methods*
  • Microtubules / metabolism*
  • Mitosis / genetics
  • Mitosis / physiology
  • Spindle Apparatus / genetics
  • Spindle Apparatus / metabolism
  • Spindle Poles / metabolism*
  • Tubulin / metabolism

Substances

  • Tubulin
  • Kinesin