Cyto-architecture constrains a photoactivation induced tubulin gradient in the syncytial Drosophila embryo

Abstract

Drosophila embryogenesis begins with nuclear division in a common cytoplasm forming a syncytial cell. Morphogen gradient molecules spread across nucleo-cytoplasmic domains to pattern the body axis of the syncytial embryo. The diffusion of molecules across the syncytial nucleo-cytoplasmic domains is potentially constrained by association with the components of cellular architecture, however the extent of restriction has not been examined so far. Here we use photoactivation (PA) to generate a source of cytoplasmic or cytoskeletal molecules in order to monitor the kinetics of their spread in the syncytial Drosophila embryo. Photoactivated PA-GFP and PA-GFP-Tubulin within a fixed anterior area diffused along the antero-posterior axis. These molecules were enriched in cortical cytoplasm above the yolk-filled center suggesting that the cortical cytoplasm is phase separated from the yolk-filled center. The length scales of diffusion were extracted using exponential fits under steady state assumptions. PA-GFP spread to greater distance as compared to PA-GFP-Tubulin. Both gradients were steeper and more restricted when generated in the center of the embryo probably due to a higher density of nucleo-cytoplasmic domains. The length scale of diffusion for PA-GFP-Tubulin gradient increased in mutant embryos containing short plasma membrane furrows and disrupted tubulin cytoskeleton. The PA-GFP gradient shape was unaffected by cyto-architecture perturbation. Taken together, these data show that PA-GFP-Tubulin gradient is largely restricted by its incorporation in the microtubule network and intact plasma membrane furrows. This photoactivation based analysis of protein spread across allows for interpretation of the dependence of gradient formation on the syncytial cyto-architecture.