Regulation of Mitotic Microtubule Dynamic Instability in Monopolar Spindles by Bundling and Kinetochore Attachment

Abstract

ObjectiveThe dynamics of mitotic microtubules are important for mitotic spindle assembly and chromosome segregation, but descriptive parameters have proved challenging to measure directly. We are studying the dynamic instability of single and bundled mitotic microtubules (MTs) that originate from the spindle pole bodies in fission yeasts. We marked the α‐tubulin with an m‐Cherry tag expressed at low levels to minimally perturb MT dynamics and labeled the kinetochores with a GFP tag. Using a temperature sensitive kinesin‐5 allele (cut7–24) at restrictive temperature (37°C) in which bipolar spindle assembly is blocked, we observe monopolar spindles whose dynamic MTs can be imaged directly.Results and ConclusionsThis persistent monopolar spindle allows the measurement of growth, shrinkage, catastrophe, and rescue rates for both single and bundled MTs. The kinetochores are dynamic and appear to move along bundled MTs allowing us to study correlated dynamics of the MTs and kinetochores. We have compared MT dynamics and bundling in this strain with others that contain additional perturbations to proteins that affect MT dynamics and bundling. To quantify these parameters, we have authored software to automate detection and measurement of MT and kinetochore dynamics and their changes over time in monopolar spindles. To quantify the rate of tubulin turnover in this system, we have performed photobleaching experiments in which we detect the substitution of unbleached tubulin dimers for bleached ones using fluorescence recovery (FRAP).Support or Funding InformationThis work was supported by NSF grants DMR‐1551095 to MDB, and NIH grants K25 GM110486 to MDB and R01 GM033787 to JRM.