The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. This requires attachment of spindle microtubules to kinetochores, specialized structures formed at centromeres. Kinetochores are more than just structural elements that mediate faithful chromosome inheritance. The unattached kinetochore is also the signal generator for the mitotic checkpoint (also known as the spindle assembly checkpoint), the major cell cycle control mechanism in mitosis. Each unattached kinetochore generates a """"""""wait anaphase"""""""" inhibitor that blocks destruction of cyclin B and securin, thereby blocking disjoining of duplicated sister chromosomes and advance into anaphase. We have previously identified a microtubule-dependent motor, CENPE, to be multifunctional these mitotic events. It directly tethers spindle microtubules to kinetochores and it functions as the cyclin-like activator of the essential mitotic checkpoint kinase BubR1. Microtubule capture by CENP-E in turn inactivates BubR1 kinase activity, thereby silencing mitotic checkpoint signaling and permitting anaphase onset. We now propose to determine the properties of CENP-E in microtubule capture at kinetochores (with electron microscopy), the motor properties of CENP-E (including processivity and residence time when microtubule bound), and how CENP-E acts to affect coupling to dynamic microtubules. The functional role of an unusual CENPE modification, farnesylation, will be determined. A major effort will use all purified components to reconstruct mitotic checkpoint signaling in vitro and to identify the """"""""wait anaphase"""""""" inhibitor(s) produced by unattached kinetochores. Moreover, recognizing that most human solid tumors are aneuploid, that is, have other than the correct number of 46 chromosomes, we will exploit our discovery that reduced levels of CENP-E generate accelerated rates of such aneuploidy to enable a test of how whole chromosomal aneuploidy affects tumorigenesis initiated by loss of each of the major tumor suppressor genes. This will provide a test of the hypothesis posed more than 100 years ago by the great cytologist Boveri that aneuploidy drives tumorigenesis. Finally, recognizing that successful cancer therapeutics such as taxol yield chronic activation of the mitotic checkpoint, we will examine the mechanisms underlying cell death or adaptation after chrom0ic mitotic checkpoint arrest.

Public Health Relevance

Unattached kinetochores control the cell cycle clock during cell division by generating a """"""""wait"""""""" signal, the mitotic checkpoint, which delays cell cycle advance until all chromosomes have successfully attached to spindle microtubules. How this signaling works will be examined by determining how a kinetochore motor protein affects chromosome attachment to spindle microtubules and acts to activate and silence an essential checkpoint kinase. The overall signaling pathway will be determined by reconstructing it from individual components. Finally, since some successful anti-tumor drugs in humans chronically activate the mitotic checkpoint, the mechanisms tha determine cell fate, cell death or adaptation, when so arrested will be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029513-31
Application #
8103935
Study Section
Nuclear Dynamics and Transport (NDT)
Program Officer
Deatherage, James F
Project Start
1981-07-01
Project End
2012-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
31
Fiscal Year
2011
Total Cost
$635,178
Indirect Cost
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Bertuzzi, Stefano; Cleveland, Don W (2015) The curious incident of the translational dog that didn't bark. Trends Cell Biol 25:187-9
Kulukian, Anita; Holland, Andrew J; Vitre, Benjamin et al. (2015) Epidermal development, growth control, and homeostasis in the face of centrosome amplification. Proc Natl Acad Sci U S A 112:E6311-20
Vitre, Benjamin; Holland, Andrew J; Kulukian, Anita et al. (2015) Chronic centrosome amplification without tumorigenesis. Proc Natl Acad Sci U S A 112:E6321-30
Topham, Caroline; Tighe, Anthony; Ly, Peter et al. (2015) MYC Is a Major Determinant of Mitotic Cell Fate. Cancer Cell 28:129-40
Holland, Andrew J; Reis, Rita M; Niessen, Sherry et al. (2015) Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors. Mol Biol Cell 26:1845-56
Han, Joo Seok; Vitre, Benjamin; Fachinetti, Daniele et al. (2014) Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling. Proc Natl Acad Sci U S A 111:E4185-93
Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier et al. (2014) Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E. Mol Biol Cell 25:2272-81
Holland, Andrew J; Cleveland, Don W (2014) Polo-like kinase 4 inhibition: a strategy for cancer therapy? Cancer Cell 26:151-3
Mirzaa, Ghayda M; Vitre, Benjamin; Carpenter, Gillian et al. (2014) Mutations in CENPE define a novel kinetochore-centromeric mechanism for microcephalic primordial dwarfism. Hum Genet 133:1023-39
Silk, Alain D; Zasadil, Lauren M; Holland, Andrew J et al. (2013) Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors. Proc Natl Acad Sci U S A 110:E4134-41

Showing the most recent 10 out of 66 publications