Analysis of cytoplasmic organization and compartmentalization in syncytial drosophila embryo

Abstract

Syncytial nuclei found in fungi plant endosperm insect embryos and muscles maintainan autonomous nuclear cytoplasmic region of influence thereby generatingcompartments within a seemingly shared cytoplasm Molecular diffusion acrosssyncytial nucleo cytoplasmic domains may be constrained by binding to differentcomponents of the cytoarchitecture The syncytial Drosophila blastoderm embryoprovides a paradigm to test the impact of binding to the cytoarchitecture on cytoplasmicdiffusion We have analyzed the organization of the cytoplasm and further tested theextent of diffusion of molecules that bind differentially to the cytoarchitecture acrossnucleo cytoplasmic domains We find that the cytoplasm is enriched in a phaseseparated manner at the cortex of the syncytial Drosophila embryo above the yolk Photobleaching analysis shows that molecules are more constrained above the nucleias compared to below the nuclei in the syncytial embryo We generated ectopicdiffusion sources of photoactivatable GFP PA GFP or PA GFP Tubulin byphotoactivation in the antero posterior axis We found that PA GFP Tubulin which isincorporated in the tubulin cytoskeleton spreads less than PA GFP in the anterior posterior direction Using anteriorly localized ectopic probes we have tested howmolecules of different sizes or with different cyto architectural interactions spread inembryo Expression of heavier fluorescent molecules at the anterior showed a shallowlinear gradient in the antero posterior axis as compared to RFP that spread uniformlythroughout the embryo Expression of anteriorly PH PLC CFP that has an attachment toplasma membrane phospholipids however created an exponential gradient We havefurther tested what cyto architectural components can restrict these gradients usingBicoid gradient as a functional readout Mutants affecting the cytoarchitecture of thesyncytial embr newline newline