All life depends on compartmentalization, starting with organisms, to cells, down to organelles within cells. In this interdisciplinary collaboration, the PIs will elucidate the rules underlying cellular compartmentalization in bacteria. While compartmentalization within cells is often facilitated by membranes, bacteria do not typically contain membrane-enclosed organelles. Instead, bacteria must rely on alternate mechanisms such as phase separation for spatial and functional organization and regulation of biochemical activity. This mechanism allows for the formation of distinct phases or domains with different structures, functions, and material properties starting from a homogeneous mixture. Recently, soft-matter theories of phase separation of liquid mixtures have tremendously advanced understanding of the biological organization. However, the bacterial cytoplasm consists of mixtures of complex, structured fluids. Their phase separation and regulation by non-equilibrium processes such as enzymatic activity are not well understood. The PIs will use data-driven mathematical modeling and state-of-the-art experiments to obtain a quantitative understanding of the formation of phase-separated condensates and their impact on the organization of genetic material and protein diffusion in bacteria. The findings will provide insights into how intracellular phase separation drives and determines cellular properties and functions, and connects genotype to phenotype. The PIs will educate and train a new generation of scientists in mathematical modeling and biology, and promote diversity in the STEM workforce. They will co-organize Biophysics workshops to stimulate interactions among scientists and industrial labs and introduce trainees to the local academic and industrial research community.

Cells use compartmentalization to create spatial organization, allowing them to carry out biochemical processes and control biomolecular structures within distinct microenvironments. This collaborative project will test the hypothesis that bacteria use intracellular phase separation to achieve compartmentalization, allowing for rapid exchange of molecules with the cytoplasm without the need for internal membranes. Upon stress, bacterial chromosomes are reorganized by the Dps protein into a tightly compacted condensate with liquid crystalline properties. To determine the biophysical mechanisms underlying Dps-DNA condensates, single-molecule fluorescence microscopy will map the phase diagram of the condensate system as a function of physiologically relevant environmental conditions. These experiments will complement active particle and continuum models that will predict the phase-separated morphologies and the degree of liquid crystallinity of the condensate. The viscoelastic and mechanical structure-function properties of the condensate will be measured via active microrheology; polarized light microscopy will identify any large ordered domains within the condensate. Mathematical approaches will determine the structural and orientational order, allowing for the construction of a microscopic model of the phase separation and phase ordering of the condensate. To evaluate the hypothesis that small molecules can diffuse rapidly within Dps:DNA condensates to promote enzymatic activities including transcription, the diffusion of molecules within condensed droplets will be directly measured by total internal reflection fluorescence microscopy. Mathematical examination of the particle trajectories will reveal the accessibility of different DNA regions and quantitatively characterize the motility of different types of biomolecules, advancing the understanding of how structure-function properties of biomolecular condensates regulate cellular activities.

This award is being co-funded by the Division of Molecular and Cellular Biosciences (MCB) through the Systems and Synthetic Biology and the Genetic Mechanisms Programs, and the MPS Division of Mathematical Sciences (DMS) through the Mathematical Biology Program.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

National Science Foundation (NSF)
Division of Mathematical Sciences (DMS)
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Zhilan Feng
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University of Rochester
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