Characterization of Dynamin Function In Cytoskeleton Reorganization and Membrane Trafficking During Early Drosophila Embryogenesis
Dynamin is a high molecular weight GTPase required for membrane fission during endocytosis at the plasma membrane and trans-Golgi network. More recently it has been found to have a role in centrosome cohesion and actin-dependent membrane and cytoskeleton remodelling. The Drosophila embryo at the early stages of development is an excellent model system to study the molecular mechanisms employed in membrane trafficking coupled with cytoskeletal rearrangement. The first 13 nuclear divisions occur with incomplete cytokinesis and the plasma membrane extends around each nucleus to give rise to individual epithelial cells only during the interphase of the 14th division. Unique temperature sensitive paralytic mutants at the single Drosophila shibire have been crucial in elucidating its role in endocytosis. To further investigate the function of dynamin in Drosophila development, transgenic flies expressing fluorescently tagged wild type and mutant Dynamin were generated. Live imaging of dynamin-GFP in the early Drosophila embryo shows localization on the plasma membrane, centrosome and spindle. Fluoresence recovery after photobleaching indicates that it is freely exchanging between these structures and the cytoplasm. A similar localization is seen for clathrin light chain (Clc) -GFP which is essential for early stages of endocytosis.
Figure: The localization pattern of Dynamin/shibire-GFP during nuclear cycle 12. Note the enrichment at the plasma membrane and the spindle.
Genetic analysis shows that Dynamin-GFP partially rescues the temperature sensitive phenotype of shibire mutants. A combination of temperature sensitive shibire mutants with various molecular markers is being used to understand the function of dynamin at early stages of cell division. The ongoing experiments include a dissection of the role of Dynamin in membrane trafficking versus cytoskeleton remodelling. These will be achieved in the following ways: 1) assess the status of molecules essential for stabilising adherens junctions; 2) critically examine the localization of binding partners of Dynamin which are necessary for F-Actin stabilization 3) identify binding partners of Dynamin in the wild type and mutant developing embryo by immunoprecipitation with anti-Dynamin antibodies. These studies seek to reveal the mechanism for the less understood role of Dynamin in cytoskeleton remodelling in cytokinesis.
Studies conducted by Richa Rikhy.