Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle.

Isabelle Loiodice,Phong Tran,Ryan Cochran,Divya Bhatt,Sebastien Schaub,Marcel Janson,Julie Czupryna,Penny Tavormina,Chuanhai Fu

doi:10.3390/biom9030086

Abstract

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

Highlights

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for investigating diverse cellular processes such as cell cycle and cell morphogenesis [1,2]
We present an in vivo measurement of the cellular αβ-tubulin concentration and define how tubulin is partitioned between soluble tubulin and MT polymer in the cell throughout the cell cycle
We previously reported that interphase and astral MT bundles are organized with their minus ends crosslinked at the cell middle or the spindle pole body (SPB), respectively, by the protein Ase1, and their dynamic distal plus ends interacting with the cell cortex [28]

Summary

Introduction

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for investigating diverse cellular processes such as cell cycle and cell morphogenesis [1,2]. Fission yeast is a good organism for quantitative dynamic imaging studies of fluorescently tagged proteins [3,4]. Fluorescence imaging has revealed the cellular concentration of actin and actin-associated proteins in fission yeast [3,4]. Similar quantifications for microtubules (MTs) and associated-proteins are lacking Processes such as MT dynamics and organization during interphase and mitosis have been dissected using fluorescent live cell imaging [5,6,7,8]. These studies described qualitatively the general organization and function of the MT cytoskeleton throughout the cell cycle.

Methods

Discussion

Conclusion