Microtubule dynamics regulation reconstituted in budding yeast lysates

• DOI: 10.1242/jcs.219386
• Publication Date: 2018-09-05T10:05:22Z
• Authors: Zane J. Bergman; Jonathan Wong; David G. Drubin; Georjana Barnes
• Keywords: Organelle; Budding

Microtubules (MTs) are important for cellular structure, transport of cargoes and segregation chromosomes organelles during mitosis. The stochastic growth shrinkage MTs, known as dynamic instability, is necessary 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 observation MTs or manipulation conditions, vitro focus on purified proteins, therefore lack complexity, cell extracts made from genetically intractable organisms. In order investigate the ensemble activities all MAPs using lysates a tractable organism, we developed cell-free assay budding yeast total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared strains expressing GFP-tubulin. polymerization pre-assembled seeds adhered coverslip was observed real time. Through use division cycle (cdc) depolymerase mutants, found that instability dependent state specific MAPs.