The importance of microtubule-dependent tension in accurate chromosome segregation.

Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.

Using Taxol-sensitized budding yeast to investigate the effect of microtubule stabilization on anaphase onset.

The microtubule (MT)-stabilizing drug Taxol (paclitaxel) is a commonly used tool to investigate MT dynamics and MT-dependent processes. Here, we present a protocol for using Taxol-sensitized budding yeast to investigate the effect of microtubule stabilization on anaphase onset. We describe steps for establishing a log phase culture, synchronizing cells in G1, arresting in metaphase, and releasing cells into Taxol. We then detail procedures for imaging and scoring anaphase onset. This protocol facilitates maintenance and reproducibility in testing drug-sensitized and Taxol-sensitized yeast strains. For complete details on the use and execution of this protocol, please refer to Proudfoot et al.1.

Checkpoint Proteins Bub1 and Bub3 Delay Anaphase Onset in Response to Low Tension Independent of Microtubule-Kinetochore Detachment.

The spindle assembly checkpoint (SAC) delays anaphase onset until sister chromosomes are bound to microtubules from opposite spindle poles. Only then can dynamic microtubules produce tension across sister kinetochores. The interdependence of kinetochore attachment and tension has proved challenging to understanding SAC mechanisms. Whether the SAC responds simply to kinetochore attachment or to tension status remains obscure. Unlike higher eukaryotes, budding yeast kinetochores bind only one microtubule, simplifying the relation between attachment and tension. We developed a Taxol-sensitive yeast model to reduce tension in fully assembled spindles. Our results show that low tension on bipolar-attached kinetochores delays anaphase onset, independent of detachment. The delay is transient relative to that imposed by unattached kinetochores. Furthermore, it is mediated by Bub1 and Bub3, but not Mad1, Mad2, and Mad3 (BubR1). Our results demonstrate that reduced tension delays anaphase onset via a signal that is temporally and mechanistically distinct from that produced by unattached kinetochores.

Discrete regions of the kinesin-8 Kip3 tail differentially mediate astral microtubule stability and spindle disassembly.

To function in diverse cellular processes, the dynamic properties of microtubules must be tightly regulated. Cellular microtubules are influenced by a multitude of regulatory proteins, but how their activities are spatiotemporally coordinated within the cell, or on specific microtubules, remains mostly obscure. The conserved kinesin-8 motor proteins are important microtubule regulators, and family members from diverse species combine directed motility with the ability to modify microtubule dynamics. Yet how kinesin-8 activities are appropriately deployed in the cellular context is largely unknown. Here we reveal the importance of the nonmotor tail in differentially controlling the physiological functions of the budding yeast kinesin-8, Kip3. We demonstrate that the tailless Kip3 motor domain adequately governs microtubule dynamics at the bud tip to allow spindle positioning in early mitosis. Notably, discrete regions of the tail mediate specific functions of Kip3 on astral and spindle microtubules. The region proximal to the motor domain operates to spatially regulate astral microtubule stability, while the distal tail serves a previously unrecognized role to control the timing of mitotic spindle disassembly. These findings provide insights into how nonmotor tail domains differentially control kinesin functions in cells and the mechanisms that spatiotemporally control the stability of cellular microtubules.