Spatial Organization of Cytoplasm Studied with Xenopus Egg Extract

Abstract

Spatial organization is essential for biological function at all scales. Within the cytoplasm, various processes require dynamic distribution of intracellular components and establishment of spatial compartments. During mitosis, cytoplasmic organelles as well as chromatin are partitioned into daughter cells; for protein quality control, aggregated proteins are sequestered into specific aggresome structures. In my thesis, I used Xenopus laevis egg extract as a model system to study spatial organization of cytoplasm in these processes. Cell division during early embryogenesis requires spatial organization of the cytoplasm on length scales of up to a millimeter in species with large eggs. Questions that remain unclear include how intracellular forces are generated at such enormous spatial scales, and how the abundant cytoplasmic components are partitioned prior to cytokinesis. Microtubules are thought of as the main cytoskeletal network that spans the cytoplasm to organize mitosis and cytokinesis, while research on actin filaments and myosin II (together known as “actomyosin”) has mostly been focused on their function at the actin-rich cortex. Whether bulk actomyosin within the cytoplasm plays any role in cell division remains poorly understood. Here we developed a cell-free system with all cytoskeletal networks that recapitulated early embryonic cleavages of the frog Xenopus laevis, and used this system to study the role of bulk actomyosin. Cell-free divisions exhibited multifaceted defects when cytoplasmic F-actin was perturbed. Bulk actomyosin played a major role in partitioning cytoplasm after mitosis. It mechanically strengthened cytoplasm and microtubule asters, at the same time developed spatial heterogeneity in both material properties and stress distribution by setting up the actin depletion zone at future cleavage plane. Microtubule asters behaved as actin-reinforced composite gels that integrated centrosomes, nuclei, and organelles during their partition after mitosis. Actomyosin contraction provided driving force for the astral composite to move away from future cleavage plane. A fluid dynamics model with contractile stress recapitulated cytoplasmic flows away from the midplane. These findings uncover a role of bulk actomyosin in global partitioning of cytoplasm in large embryonic cells and lead to a novel framework for understanding cytoplasm as a composite gel whose dynamics are governed by principles of active fluid mechanics. Beyond cell division, the second part of my thesis studied how protein aggregates are selectively transported to the aggresome to maintain protein homeostasis. Using the more conventional CSF egg extract, we reconstituted MTOC-directed aggregate transport in Xenopus egg extract. High-resolution single-particle tracking revealed that dynein-mediated aggregate transport was highly episodic, with average velocity positively correlating with aggregate size. We propose that size selectivity in this process enables efficient intracellular transport of protein aggregates.