The long term goal of this project is to elucidate the in vivo function of microtubule proteins in mitosis and cellular morphogenesis. The specific approach makes use of the model system, Drosophila melanogaster, and utilizes biochemical, molecular biological, and genetic analyses to elucidate the in vivo functions of proteins characterized in vitro. This application focuses on one member of each of two classes of proteins, microtubule-associated proteins and microtubule-motility proteins. The first molecule that we will study is the 205K microtubule-associated protein, which is a constituent of the mitotic spindle and interphase cytoskeleton. The in vivo function of the 205K MAP in mitosis and morphogenesis will be determined by phenotypic analysis of 205K MAP mutants. In addition, if we find that the 205K MAP has a role in microtubule function in mitosis or morphogenesis, we will probe its role in detail through the use of in vitro mutagenesis. The second protein that we will analyze is kinesin. Kinesin is a molecule that generates microtubule movement in vitro, and which current evidence suggests is a mitotic spindle component and an element of the axonal transport system. The in vivo function of kinesin will primarily be studied by analysis of kinesin mutants, and by inhibition of kinesin expression in cultured cells using inducible expression of anti-sense kinesin RNA. To develop sufficient understanding of kinesin so that mutations can be generated in vitro and studied in vivo, and to identify domains required for in vitro functions, we will also analyze the structure and function of kinesin by sequencing and deletion analysis. To elucidate the function of other mitotic spindle components, we will use genetic analysis to study new mitotic spindle proteins. These new proteins will be identified by three methods: a) production and analysis of monoclonal antibodies; b) nucleotide homology to existing genes; and c) homology to consensus elements of existing genes such as those encoding microtubule- binding regions and nucleotide binding regions. Genetic and phenotypic analysis of mutations disrupting these proteins can then be used to study their in vivo roles. Elucidation of the in vivo functions of the 205K MAP, kinesin, and other mitotic spindle proteins will provide considerable information about the mechanisms of mitosis and cellular morphogenesis, and may shed light on human diseases such as cancer and birth defects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035252-06
Application #
3287683
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1985-08-30
Project End
1991-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Gunawardena, Shermali; Yang, Ge; Goldstein, Lawrence S B (2013) Presenilin controls kinesin-1 and dynein function during APP-vesicle transport in vivo. Hum Mol Genet 22:3828-43
Reis, Gerald F; Yang, Ge; Szpankowski, Lukasz et al. (2012) Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila. Mol Biol Cell 23:1700-14
Falzone, Tomás L; Gunawardena, Shermali; McCleary, David et al. (2010) Kinesin-1 transport reductions enhance human tau hyperphosphorylation, aggregation and neurodegeneration in animal models of tauopathies. Hum Mol Genet 19:4399-408
Abe, Namiko; Almenar-Queralt, Angels; Lillo, Concepcion et al. (2009) Sunday driver interacts with two distinct classes of axonal organelles. J Biol Chem 284:34628-39
Shah, Sameer B; Nolan, Rhiannon; Davis, Emily et al. (2009) Examination of potential mechanisms of amyloid-induced defects in neuronal transport. Neurobiol Dis 36:11-25
Falzone, Tomás L; Stokin, Gorazd B; Lillo, Concepción et al. (2009) Axonal stress kinase activation and tau misbehavior induced by kinesin-1 transport defects. J Neurosci 29:5758-67
Stokin, Gorazd B; Almenar-Queralt, Angels; Gunawardena, Shermali et al. (2008) Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides. Hum Mol Genet 17:3474-86
Xia, Chun-Hong; Roberts, Elizabeth A; Her, Lu-Shiun et al. (2003) Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A. J Cell Biol 161:55-66
Gunawardena, Shermali; Her, Lu-Shiun; Brusch, Richard G et al. (2003) Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron 40:25-40
Ji, Jun-Yuan; Haghnia, Marjan; Trusty, Cory et al. (2002) A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability. Genetics 162:1179-95

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