Analysis of cytoplasmic organization and compartmentalization in syncytial Drosophila embryo

Abstract

Syncytial nuclei found in fungi, plant endosperm, insect embryos and muscles maintain an autonomous 'nuclear-cytoplasmic region of influence', thereby generating compartments within a seemingly shared cytoplasm. Molecular diffusion across syncytial nucleo-cytoplasmic domains may be constrained by binding to different components of the cytoarchitecture. The syncytial Drosophila blastoderm embryo provides a paradigm to test the impact of binding to the cytoarchitecture on cytoplasmic diffusion. We have analyzed the organization of the cytoplasm and further tested the extent of diffusion of molecules that bind differentially to the cytoarchitecture across nucleo-cytoplasmic domains. We find that the cytoplasm is enriched in a phase separated manner at the cortex of the syncytial Drosophila embryo above the yolk. Photobleaching analysis shows that molecules are more constrained above the nuclei as compared to below the nuclei in the syncytial embryo. We generated ectopic diffusion sources of photoactivatable-GFP (PA-GFP) or PA-GFP-Tubulin by photoactivation in the antero-posterior axis.We found that PA-GFP-Tubulin which is

incorporated in the tubulin cytoskeleton, spreads less than PA-GFP in the anterior- posterior direction. Using anteriorly localized ectopic probes,we have tested how

molecules of different sizes or with different cyto-architectural interactions spread in embryo. Expression of heavier fluorescent molecules at the anterior showed a shallow linear gradient in the antero-posterior axis as compared to RFP that spread uniformly throughout the embryo. Expression of anteriorly PH-PLC-CFP that has an attachment to plasma membrane phospholipids however, created an exponential gradient. We have further tested what cyto-architectural components can restrict these gradients, using Bicoid gradient as a functional readout. Mutants affecting the cytoarchitecture of the syncytial embryo differentially affect these gradients and our study proposes a role of the cytoarchitecture in differentially constraining gradients based on their molecular properties.