A Mechanistic Study of Complex Biological Phenomena: From Membrane-less Organelle Assembly to Cell Cycle Regulation

Abstract

This work is an effort to disentangle different mechanisms that drive complex biological phenomena, from the assembly of membrane-less organelles and their spatiotemporal regulation to the regulation of cell cycle events. To do so, we have taken advantage of the differential sensitivity of distinct mechanisms to temperature by utilizing a microfluidics-based temperature assay, in combination with quantitative live imaging and genetic approaches, in early Drosophila embryos. Our results indicate that the quintessential membrane-less organelle, the nucleolus, forms through two independent mechanisms, namely active assembly and thermodynamically-driven phase separation. These two independent mechanisms are coordinated by rDNA to ensure that the organelle forms at the right time and the right place. Transcription of rRNA spatiotemporally regulates the phase separation by overcoming the initial nucleation step in the formation of these assemblies. rDNA is also necessary for the formation of active assemblies, and therefore can be considered as a common coordinator of the active and thermodynamic mechanisms of the nucleolus assembly. Finally, by using a similar temperature-base assay we are able uncouple various cell cycle events and show that while entry into prophase can occur independent of CDK1 activity, initiation of prometaphase always coincides with the activation of CDK1.