Microtubule dynamics regulation reconstituted in budding yeast lysates

Area of Science:

• Cell Biology
• Biophysics

Background:

• Microtubules (MTs) are essential cytoskeletal components involved in cell structure, intracellular transport, and chromosome segregation during mitosis.
• The dynamic instability of MTs, characterized by stochastic growth and shrinkage, is critical for their diverse cellular functions.
• Previous research on microtubule-associated proteins (MAPs) faced limitations due to either the complexity of in vivo studies or the lack of cellular context in in vitro assays using purified proteins or extracts from non-tractable organisms.

Purpose of the Study:

• To develop and validate a novel cell-free assay using budding yeast lysates to investigate the collective effects of all MAPs on MT dynamics.
• To examine the real-time polymerization and dynamic instability of microtubules in a cellular context without genetic manipulation of individual MAPs.
• To determine the influence of cell cycle state and specific MAP activities on MT polymerization and dynamic instability.

Main Methods:

• Development of a cell-free assay utilizing total internal reflection fluorescence (TIRF) microscopy with lysates from yeast strains expressing GFP-tubulin.
• Observation of MT polymerization from pre-assembled MT seeds adhered to a coverslip in real time.
• Utilized cell division cycle (cdc) and MT depolymerase mutants to perturb cellular states and analyze their effects on MT dynamics.

Main Results:

• Successfully established a functional cell-free system for observing MT dynamics in budding yeast lysates.
• Demonstrated that MT polymerization and dynamic instability are significantly influenced by the cell cycle stage.
• Identified the dependence of MT dynamics on the collective activities of MAPs present in the yeast lysate.

Conclusions:

• The developed cell-free assay provides a powerful tool to study the ensemble activities of MAPs on MT dynamics in a genetically tractable organism.
• MT polymerization and dynamic instability are regulated by the cell cycle and the integrated functions of various MAPs.
• This approach overcomes limitations of previous methods, enabling a more comprehensive understanding of MT regulation within a cellular context.