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 kinetochore-bound microtubule-dependent motor, CENP-E;to be the target of an Aurora kinase- protein phosphatase I (PP1) switches that regulates congression of initially misoriented chromosomes. We will now determine how this switch modulates CENP-E motor activity and the role of PP1-bound CENP-E in reactivating capture of spindle microtubules by kinetochores of initially misaligned, polar chromosomes. A major effort will use all purified components to reconstruct mitotic checkpoint signaling, and with this we will determine the mechanism for its selective inhibition of ubiquitination of cyclin B and securin, as well as the roles played by essential checkpoint kinases. Centrosomes are the major microtubule organizing centers of animal cells and play a particularly important role during mitosis, where they form the poles of the bipolar microtubule spindle upon which chromosomes are segregated. The duplication of the centrosome is tightly controlled, and extra centrosomes can cause errors in spindle formation that lead to subsequent chromosome missegregation. Supernumerary centrosomes are present in many types of cancers and can be found in early premalignant lesions. More than 100 years ago the great cytologist Boveri proposed a link between tumorigenesis and aneuploidy, including the still untested contribution of centrosome amplification. Using a conditional mouse model(s) in which extra centrosomes can be induced, we will determine whether centrosome amplification 1) promotes cellular transformation, 2) the formation of spontaneous tumors, 3) is capable of facilitating the development of carcinogen-induced tumors, and 4) is able to accelerate the development (or increase the aggressiveness or metastatic potential) of tumors driven by the loss of a tumor suppressor gene. Finally, we will utilize gene-targeting and replacement in cultured human cells to determine how a key upstream regulator of centrosome duplication, Polo-like kinase 4, acts to limit centrosome duplication to once per cell cycle.

Public Health Relevance

The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. An abnormal chromosome number, aneuploidy, has long been linked to human tumors. The effort here will identify key steps through which the major cell cycle mechanism in mitosis, the mitotic checkpoint, acts to prevent chromosome missegregation. Additionally, amplification of centrosomes, the microtubule organizing centers of mitotic spindles, has also long been associated with tumorigenesis. Using mice in which extra centrosome replication can be induced, it will be determined whether centrosome amplification promotes cellular transformation, the formation of spontaneous tumors and accelerates the development of tumors that develop from loss of a tumor suppressor gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029513-33
Application #
8517730
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Deatherage, James F
Project Start
1981-07-01
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
33
Fiscal Year
2013
Total Cost
$585,554
Indirect Cost
$226,098
Name
Ludwig Institute for Cancer Research Ltd
Department
Type
DUNS #
627922248
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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
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
Gudimchuk, Nikita; Vitre, Benjamin; Kim, Yumi et al. (2013) Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips. Nat Cell Biol 15:1079-88
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
Han, Joo Seok; Holland, Andrew J; Fachinetti, Daniele et al. (2013) Catalytic assembly of the mitotic checkpoint inhibitor BubR1-Cdc20 by a Mad2-induced functional switch in Cdc20. Mol Cell 51:92-104
Gassmann, Reto; Holland, Andrew J; Varma, Dileep et al. (2010) Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells. Genes Dev 24:957-71
Cetin, Bulent; Cleveland, Don W (2010) How to survive aneuploidy. Cell 143:27-9
Kim, Yumi; Holland, Andrew J; Lan, Weijie et al. (2010) Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E. Cell 142:444-55
Holland, Andrew J; Lan, Weijie; Cleveland, Don W (2010) Centriole duplication: A lesson in self-control. Cell Cycle 9:2731-6

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