Georjana Barnes

Ch-TOG family proteins, including the budding yeast Stu2, are essential for spindle formation and chromosome segregation. Such functions depend on an array of activities ranging from microtubule nucleation, polymerization, and depolymerization to conferring tension sensitivity to kinetochores. This functional diversity makes it challenging to dissect these various functions and understand their relative importance. Here, we developed separation-of-function mutants and used artificial tethering tools to elucidate several important mechanistic insights into Stu2’s essential role. We show that Stu2’s microtubule polymerization activity depends on its basic linker region but is surprisingly dispensable for viability; that in fact, Stu2 carries out an essential kinetochore-associated function; and finally, that Stu2’s precise location within the kinetochore is critical for its function, suggesting a spatial separation mode of action may underlie its ability to confer tension sensitivity. Our findings highlight the significance of Stu2’s kinetochore role and provide insights into the molecular mechanisms by which it performs its various functions.

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae . Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.

The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and cytoplasmic microtubules (MTs) used to segregate chromosomes remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has unique roles in both compartments and has been implicated in capturing unattached kinetochores, stabilizing crosslinked interpolar microtubules (MT), and creating intranuclear and cytoplasmic MT arrays at the spindle pole body for kinetochore capture and karyogamy, respectively. Here, we show that two proteins, Cik1 and Vik1, that form heterodimers with Kar3, regulate its localization and function within the cell and along MTs in a cell cycle-dependent manner. Using a cell cycle synchronized, yeast MT dynamics reconstitution assay in cell lysate, we found that Kar3Vik1 induces MT catastrophes in S phase and metaphase and limits MT polymerization in G1 and anaphase. In contrast, Kar3Cik1 is a catastrophe and pause promoter in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track single-molecules, we saw that Kar3Cik1 is necessary for tracking MT plus-ends in S phase and metaphase, but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally. SUMMARY STATEMENT: We show through biochemical reconstitution experiments and live-cell imaging that the functions and localization of the budding yeast kinesin-14, Kar3, are dictated by which of its two accessory protein binding partners, Cik1 or Vik1, it binds to and by the cell cycle stage.

Ch-TOG family proteins, including the budding yeast Stu2, are essential for spindle formation and chromosome segregation. Such functions depend on an array of activities ranging from microtubule nucleation, polymerization and depolymerization, to conferring tension sensitivity to kinetochores. This functional diversity makes it challenging to dissect these various functions and understand their relative importance. Here, we developed separation-of-function mutants and used artificial tethering tools to elucidate several important mechanistic insights into Stu2’s essential role. We show that Stu2’s microtubule polymerization activity depends on its basic linker region but is surprisingly dispensable for viability; that in fact, Stu2 carries out an essential kinetochore-associated function; and finally, that Stu2’s precise location within the kinetochore is critical for its function, suggesting a spatial separation mode of action may underlie its ability to confer tension sensitivity. Our findings highlight the significance of Stu2’s kinetochore role and provide insights into the molecular mechanisms by which it performs its various functions.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between endogenously tagged tubulin, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae . Using both TIRF microscopy and cryo-correlative light microscopy and electron tomography, we successfully reconstituted microtubule-bound, intact kinetochores. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.

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 focus either 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 total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared from yeast strains expressing GFP-tubulin. 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 on the cell cycle state and the activities of specific MAPs.

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly.

We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and we quantified recruitment in fixed cells. Following induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres following kinetochore assembly. Following their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores, and that lateral attachment licenses Mad1 removal from kinetochores, following a kinetic delay.