The overall objective of this research program is to determine the molecular and structural mechanisms which regulate microtubule (MT) and spindle assembly dynamics and the mechanisms that move chromosomes. A major strength of our application is the development and application of new techniques in quantitative optical microscopy, fluorescent analog cytochemistry, caged compounds, laser optical traps and scissors, video- and digital imaging to measure the dynamics of individual MTs and associated processes in living cells and reconstituted preparations in vitro. The major specific aims are: (1) develop new light microscopy methods for tracking the motion of objects like kinetochores and microtubule ends, optical traps for measuring forces and manipulating objects relative to MTs, digital imaging methods for photon limited analysis of molecular and structural dynamics for spindles in tissue cells, extracts and in genetic organisms like yeast and Drosophila; and photoactivation techniques for GFP and other caged fluorophores and compounds for analysis of spindle and MT dynamics: (2) use high resolution video and digital microscopy assays of purified and cell extract systems to determine the molecular basis of MT dynamic instability, identify cell cycle dependent regulatory factors (non motor and MT motors) and obtain tubulin peptide ligands from phage-displayed random peptide libraries; (3) employ laser optical traps, laser scissors and calibrated glass microneedles to measure MT mechanical properties, force generation by MT polymerization/depolymerization, the strength of MT tip attachment molecules (TACs or couplers), kinetochore forces, pole-complex forces and polar ejection forces on chromosome arms and MT motor activity; (4) use fluorescence photoactivation approaches to determine the functions and dynamics of the molecular components of spindle fibers, kinetochores and spindle pole-complexes during congression and anaphase in vertebrate tissue cells, plant cells and cytoplasmic extracts; (5) analyze the motility of minus kinesins and their function in spindle pole organization and poleward force generation; (6) use high resolution digital imaging of genetically manipulated Drosophila and yeast cells to determine the functions of different proteins in mitotic spindle dynamics, chromosome segregation and cell cycle regulation using Drosophila and yeast cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024364-19
Application #
2391843
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1978-09-01
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
19
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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
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:
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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