The overll objective of this project is to determine the molecular and structural mechanisms which regulate microtubule assembly and move chromosomes. Our approach continues to be the development of an isolated mitotic spindle preparation from sea urchin eggs which permits direct analysis of spindle structure, composition, and function. We are also focusing now on the hypothesis that calcium regulates spindle assembly and function in vivo, and that membranes endogenous to the mitotic apparatus locally control intraspindle calcium ion (CA++) concentrations. We have developed a technique which uses an EGTA lysis buffer for isolating and storing membrane-free mitotic spindles that are labile to micromolar CA++. For the EGTA-isolated spindles we intend to continue analyzing the protein composition, ultrastructure, microtubule polarity, and the mechanism by which calcium depolymerizes the microtubules and causes shortening of the spindle fibers. We are investigating optimum buffer conditions for reactivating life-like assembly-disassembly characteristics in the EGTA-isolated spindles and will isolate egg tubulin to use in the reassembly buffers. Tubulin that has been fluorescently labelled with dichlorotriazinyl fluorescein (DTAF) will help us to localize sites of tubulin incorporation and dissociation from the microtubules of EGTA-isolated spindles. We will investigate the incorporation and flux of tubulin in living spindles by microinjecting DTAF-tubulin into mitotic cells. We also intend to isolate mitotic apparatuses that retain the normal structure of the intraspindle membranes, and we will study their ultrastructure and protein composition (comparing them to the membrane-free EGTA-isolated spindles), as well as their ability to sequester CA++. The role of the membranes in regulating intraspindle CA++ concentrations will be investigated in vivo by measuring intracellular CA + fluctuations by CA++ selective microelectrodes, by monitoring changes in chlorotetracycline fluorescence, or aequorin luminescence. Our observations will be made with various microscopy techniques (polarization, darkfield, fluorescence, etc.) and an image-intensified video time-lapse recording system. Ultimately, we hope to achieve an isolated mitotic model system which, with appropriate buffer conditions, will enable us to reactivate life-like chromosome movements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024364-07
Application #
3272234
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-09-01
Project End
1986-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
7
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Suzuki, Aussie; Long, Sarah K; Salmon, Edward D (2018) An optimized method for 3D fluorescence co-localization applied to human kinetochore protein architecture. Elife 7:
Suzuki, Aussie; Gupta, Amitabha; Long, Sarah K et al. (2018) A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation. Curr Biol 28:2697-2704.e3
Salmon, Edward D; Bloom, Kerry (2017) Tension sensors reveal how the kinetochore shares its load. Bioessays 39:
Lera, Robert F; Potts, Gregory K; Suzuki, Aussie et al. (2016) Decoding Polo-like kinase 1 signaling along the kinetochore-centromere axis. Nat Chem Biol 12:411-8
Suzuki, Aussie; Badger, Benjamin L; Haase, Julian et al. (2016) How the kinetochore couples microtubule force and centromere stretch to move chromosomes. Nat Cell Biol 18:382-92
Suzuki, Aussie; Badger, Benjamin L; Salmon, Edward D (2015) A quantitative description of Ndc80 complex linkage to human kinetochores. Nat Commun 6:8161
Suzuki, Aussie; Badger, Benjamin L; Wan, Xiaohu et al. (2014) The architecture of CCAN proteins creates a structural integrity to resist spindle forces and achieve proper Intrakinetochore stretch. Dev Cell 30:717-30
Varma, Dileep; Chandrasekaran, Srikripa; Sundin, Lynsie J R et al. (2012) Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore-microtubule attachment. Nat Cell Biol 14:593-603
Wan, Xiaohu; Cimini, Daniela; Cameron, Lisa A et al. (2012) The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells. Mol Biol Cell 23:1035-46
Lawrimore, Josh; Bloom, Kerry S; Salmon, E D (2011) Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome. J Cell Biol 195:573-82

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