We will study the mechanisms of chromosome motion in the fission yeast, Schizosaccharomyces pombe. S. pombe has several virtues for mitotic research. It contains only three, easily visualized chromosomes. As in many other eukaryotes, each of its centromeres is large, comprising many l0s of kb of DNA, and each binds 2-4 microtubules (MTs). Its mitotic cycle includes prometaphase congression to the spindle equator and both anaphase A and B. Of the 10 MT-associated motors now known in this organism, we have selected 3 kinesin-like proteins (KLPs) for detailed study. One of these (Klp2p) localizes to spots on mitotic chromosomes that are probably kinetochores, and two of them (Klp5p and 6p) are essential for normal prometaphase chromosome motion and spindle length control. Our plans include four specific aims.
Aim 1) To characterize the motility and domain structure of these KLPs, using a combination of molecular, biochemical, and cell biological approaches. The results should clarify motor directionality, speed, and enzymology. They should also identify any effects of motor action on MT dynamics, characterize the protein domains responsible for cargo binding and localization, and isolate additional alleles of each motor.
Aim 2) To identify genes and their products that interact with these motors, both to bind them to cargo and to control their activity in vivo.
Aim 3) To study the structure of kinetochores in fission yeast, using electron microscope tomography and EM immunolocalization of known kinetochore components.
Aim 4) To take advantage of a recently developed method for isolating fission yeast chromosomes and characterize kinetochore-MT interaction in vitro. We will employ already-useful systems for the assembly of MTs into organized arrays and use laser tweezers to manipulate the kinetochore-MT connection. Chromosomes prepared from strains lacking specific kinetochore components and/or with chemistry modified in vitro (e.g., through the action of relevant phosphatases or kinases), will be used to identify the molecules necessary for kinetochore-MT interaction and the ways in which mechanical activities at this interface (e.g., tension) modify the physiology of the kinetochore
| McIntosh, J Richard; O'Toole, Eileen; Morgan, Garry et al. (2018) Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments. J Cell Biol 217:2691-2708 |
| Giddings Jr, Thomas H; Morphew, Mary K; McIntosh, J Richard (2017) Preparing Fission Yeast for Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Blackwell, Robert; Edelmaier, Christopher; Sweezy-Schindler, Oliver et al. (2017) Physical determinants of bipolar mitotic spindle assembly and stability in fission yeast. Sci Adv 3:e1601603 |
| Morphew, Mary K; Giddings Jr, Thomas H; McIntosh, J Richard (2017) Immunolocalization of Proteins in Fission Yeast by Electron Microscopy. Cold Spring Harb Protoc 2017: |
| Blackwell, Robert; Sweezy-Schindler, Oliver; Edelmaier, Christopher et al. (2017) Contributions of Microtubule Dynamic Instability and Rotational Diffusion to Kinetochore Capture. Biophys J 112:552-563 |
| Morphew, Mary K; Giddings Jr, Thomas H; McIntosh, J Richard (2017) Cryoelectron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017: |
| McIntosh, J Richard; Morphew, Mary K; Giddings Jr, Thomas H (2017) Electron Microscopy of Fission Yeast. Cold Spring Harb Protoc 2017: |
| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
| McIntosh, J Richard (2016) Mitosis. Cold Spring Harb Perspect Biol 8: |
| McIntosh, J Richard; Hays, Thomas (2016) A Brief History of Research on Mitotic Mechanisms. Biology (Basel) 5: |
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