Spatial organization of living systems
Living cells are composed of a complex mixture of macromolecules. To regulate their activity, cells partition these molecules into specialized compartments called organelles. Typically, membranes form a selective barrier between organelles and the cytoplasm, allowing each compartment to maintain a distinct biochemical composition that is tailored to its function. However, cells also contain organelles that are not enclosed by membranes. Instead, these dynamic compartments consist of local concentrations of proteins and nucleic acids which rapidly exchange with the surrounding cytoplasm or nucleoplasm. This observation raises a fundamental question: how do these unusual organelles assemble and stably persist without a membrane holding their components together? Furthermore, how do membraneless organelles carry out their biological functions and how is their activity regulated in response to changes in developmental and environmental conditions? Our lab seeks to answer these questions by examining two complementary model organisms: the multicellular nematode, Caenorhabditis elegans, and the quintessential bacterium, Escherichia coli.
Nonequilibrium phase separation
The nucleolus is a large membraneless organelle that produces ribosomes, molecular machines essential for cell growth and size homeostasis. It is composed of hundreds of proteins and RNAs that concentrate around ribosomal DNA sites in the nucleus. In developing C. elegans embryos, the bulk concentration of nucleolar components determines whether nucleoli assemble and, if so, their size. These observations correspond to the hallmarks of first-order phase transitions, suggesting that nucleoli form by phase separation of the nucleoplasm. Given the ubiquity of membraneless organelles in the nucleoplasm and cytoplasm, phase separation may represent a general mechanism for compartmentalization of the cell.
Classical models of phase separation, which assume that a system is at thermodynamic equilibrium, can predict nucleolar behavior in vivo. However, transcription and other metabolic processes that consume ATP drive the cell far from equilibrium. We are investigating how nonequilibrium biological activity affects intracellular phase separation and whether organisms can regulate these processes to control nucleolar assembly in response to developmental and environmental conditions.
Coordination of growth and size across length scales
When an organism grows, it must coordinate the growth and size of its internal structures. While signal transduction pathways are known to control organ and body size, the mechanisms regulating size at the cellular and subcellular levels remain poorly understood. Phase separation inherently couples the size of a membraneless organelle to the size of its container. For example, at a fixed concentration of components, nucleolar size scales directly with nuclear size such that small nuclei have small nucleoli while large cells have large nucleoli. This coupling may have important consequences for higher-order structures, as nucleolar size correlates with ribosome biogenesis and ultimately affects cell growth and size homeostasis. We are characterizing the scaling relationships between multiple levels of biological organization in order to determine whether phase separation-mediated nucleolar assembly can functionally coordinate size across length scales.
Spatiotemporal regulation of transcription
Bacteria typically lack membrane-bound organelles, so phase separation could provide an alternate strategy for functional organization of these cells. To explore whether intracellular phase separation is evolutionarily conserved across domains, we are analyzing the spatiotemporal distribution of RNA polymerase in prokaryotes. At slow growth rates, RNA polymerase is approximately uniformly distributed throughout the E. coli nucleoid. However, at fast growth rates, it concentrates into distinct puncta that colocalize with ribosomal RNA genes. These "transcriptional foci" may represent bacterial nucleoli, accelerating ribosome production in response to demand. We are determining whether the same thermodynamic forces that drive nucleolar assembly also govern the formation of transcriptional foci.
Thanks to our funding sources for making this research possible!