Microtubule dynamics regulation reconstituted in budding yeast lysates

Abstract

Microtubules (MTs) are important for cellular structure, transport of cargoes, and segregation of chromosomes and organelles during mitosis. The stochastic growth and shrinkage of MTs, known as dynamic instability, is necessary for these functions. Previous studies to determine how individual MT-associated proteins (MAPs) affect MT dynamics have been performed either through in vivo studies, which provide limited opportunity for observation of individual MTs or manipulation of conditions, or in vitro studies, which either focus on purified proteins, and therefore lack cellular complexity, or on cell extracts made from genetically intractable organisms. In order to investigate the ensemble activities of all MAPs on MT dynamics using lysates made from a genetically tractable organism, we developed a cell-free assay for budding yeast lysates using TIRF microscopy. Lysates were prepared from GFP-tubulin-expressing yeast strains and MT polymerization from pre-assembled MT seeds adhered to a coverslip was observed in real time. Through use of cell division cycle (cdc) and MT depolymerase mutants, we found that MT polymerization and dynamic instability are dependent upon the cell cycle state and the activities of specific MAPs.