Our long range goals are to understand, on the molecular level, the mechanism of cytoplasmic myosin function in vitro and in vivo and to determine which cellular movements require myosin for force production. We focus on the function of cytoplasmic myosin in Drosophila for two reasons. First, a diverse array of interesting movements provide the structural basis for spatial differentiation of the embryo and are characteristic of various movements seen in other cells. They include nuclear divisions and migrations, pole cell formation and cellularization, and the complex cellular migrations and shape changes of gastrulation. Moreover, these movements are amenable to study by a range of powerful approaches. We used classical protein biochemical and immunological methods to purify and characterize cytoplasmic myosin from Drosophila cells in culture. This protein is myosin by five structural and three functional criteria. It is distinct from the muscle myosin heavy chain isoform by peptide mapping and immunochemical criteria. The real advantage of this organism is of course its accessibility to genetic, molecular biological and modern molecular genetic manipulation. We used antibodies to screen a library of Drosophila genomic DNA in the expression vector lambda gtll and have isolated a fragment of the gene that encodes cytoplasmic myosin. Its gene product shares epitopes with cytoplasmic but not muscle myosin heavy chain and hybridizes with a 7.4 kb message that is sufficiently large to encode this 205 kD polypeptide. Hybridization studies with restriction fragments of Drosophila DNA and clones that encode muscle myosin heavy chain confirm that it is not part of the muscle myosin gene. Moreover, preliminary mapping studies by in situ hybridization with polytene chromosomes show that this gene maps to 60E,F, distinct from the location of the muscle myosin heavy chain gene. Here we propose a multidisciplinary approach to the analysis of cytoplasmic myosin that includes further characterization of cytoplasmic myosin function in vitro, localization of this protein in embryos and cells, the use of site specific antibodies to evaluate myosin function in vitro and in vivo, characterization of the gene that encodes this peptide and its transcription unit, synthesis of cytoplasmic myosin heavy chain or its fragments in bacterial and eukaryotic expression vector systems for detailed analysis of protein structure/function relationships in vitro, and genetic manipulation of cytoplasmic myosin in living cells and embryos by cultured cell transformation and P-element mediated germ line transformation. Together these studies forge a comprehensive investigation of the mechanism of cytoplasmic myosin function and its role in cellular movements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033830-07
Application #
3283912
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1984-09-30
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
7
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
Mortensen, Richard D; Moore, Regan P; Fogerson, Stephanie M et al. (2018) Identifying Genetic Players in Cell Sheet Morphogenesis Using a Drosophila Deficiency Screen for Genes on Chromosome 2R Involved in Dorsal Closure. G3 (Bethesda) 8:2361-2387
Lo, Wei-Chang; Madrak, Craig; Kiehart, Daniel P et al. (2018) Unified biophysical mechanism for cell-shape oscillations and cell ingression. Phys Rev E 97:062414
Aristotelous, A C; Crawford, J M; Edwards, G S et al. (2018) Mathematical models of dorsal closure. Prog Biophys Mol Biol 137:111-131
Guo, Yuting; Li, Di; Zhang, Siwei et al. (2018) Visualizing Intracellular Organelle and Cytoskeletal Interactions at Nanoscale Resolution on Millisecond Timescales. Cell 175:1430-1442.e17
Kiehart, Daniel P; Crawford, Janice M; Aristotelous, Andreas et al. (2017) Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System. Annu Rev Cell Dev Biol 33:169-202
Cao, Jingli; Wang, Jinhu; Jackman, Christopher P et al. (2017) Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue. Dev Cell 42:600-615.e4
Lu, Heng; Sokolow, Adam; Kiehart, Daniel P et al. (2016) Quantifying dorsal closure in three dimensions. Mol Biol Cell 27:3948-3955
Marston, Daniel J; Higgins, Christopher D; Peters, Kimberly A et al. (2016) MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation. Curr Biol 26:2079-89
Goldstein, Bob; Kiehart, Daniel P (2015) Moving Inward: Establishing the Mammalian Inner Cell Mass. Dev Cell 34:385-6
Lu, Heng; Sokolow, Adam; Kiehart, Daniel P et al. (2015) Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila. Biophys J 109:2406-17

Showing the most recent 10 out of 66 publications