Quality control and spatial regulation of bacterial condensates - PROJECT SUMMARY/ABSTRACTS Condensates play a crucial role in spatially organizing and regulating biochemical reactions. Dynamic condensates arise from phase separation, while insoluble aggregates result from protein misfolding. Although both can manifest as fluorescent foci in bacterial cells, they exhibit distinct biophysical properties and cellular functions. Overcoming the challenge of probing the material state of these nanometer-sized foci in small bacterial cells, I developed an experimental framework to assess phase separation in bacteria during my postdoctoral training. My framework also challenges the paradigm that inclusion bodies are solid aggregates, and calls for a thorough re-examination of processes primarily studied with inclusion bodies, such as protein quality control (PQC). In the same study, I demonstrated that the material state of protein complexes indeed influences their association with chaperones. While bacterial inclusion bodies are nucleoid-excluded and localize at the cell poles, functional condensates can be dynamically positioned on the nucleoid, as observed with carboxysomes. Carboxysomes are bacterial microcompartments responsible for CO2 fixation. The carboxysome positioning system represents the only known system for condensate positioning and the governing mechanism is still unclear. Therefore, this proposal for a K99/R00 Pathway to Independence seeks to determine how the material state of protein complexes influences mechanisms of PQC (Aim 1), and to develop minimal positioning systems for studying and manipulating condensates in bacteria (Aim 2). The outcomes of this work will advance our understanding of the differences in PQC principles governing bacterial condensates and aggregates, and contribute to determining the mechanism(s) of spatial regulation of bacterial condensates. These contributions are expected to lay a robust foundation for the development of therapeutics targeted at condensates and tools to spatially organize them. Building on the systems and methods developed in this proposal, my long-term goal is to establish an independent research group investigating (i) how the material state of protein complexes influences cellular processes that condensates are involved in, such as cell division and pathogenesis, and (ii) investigating the formation, functions, and spatial regulations of condensates and bacterial microcompartments from uncultivable bacteria in the human microbiome. The proposed training will provide me with the additional scientific expertise, including technical training in cell-free reconstitution and live-cell single-molecule tracking. This award will further my professional development including formal training in laboratory management, grant writing, and a tailored plan to support my applications to faculty positions. The University of Michigan, with its exemplary scientific and professional environment, is an ideal setting to support the training outlined in this proposal and ensure my success in launching an independent research program.
