Collaborative Research: MODULUS:Decoding the Rules of Phase Separation in Bacterial Chromatin

所有生命都依赖于区隔化，从有机体到细胞，再到细胞内的细胞器。在这个跨学科的合作中，pi将阐明细菌细胞区隔化的规则。虽然细胞内的区隔化通常由膜促进，但细菌通常不含有膜封闭的细胞器。相反，细菌必须依靠相分离等替代机制来进行空间和功能组织以及生化活动的调节。这种机制允许从均匀混合物开始形成具有不同结构，功能和材料特性的不同相或域。近年来，液体混合物相分离的软物质理论极大地促进了对生物组织的认识。然而，细菌的细胞质是由复杂的、有结构的液体混合物组成的。它们的相分离和非平衡过程（如酶活性）的调节尚不清楚。pi将使用数据驱动的数学模型和最先进的实验来定量了解相分离凝聚物的形成及其对细菌中遗传物质组织和蛋白质扩散的影响。这些发现将为细胞内相分离如何驱动和决定细胞特性和功能以及将基因型与表型联系起来提供见解。pi将在数学建模和生物学方面教育和培训新一代科学家，并促进STEM劳动力的多样性。他们将共同举办生物物理学研讨会，以促进科学家和工业实验室之间的互动，并将学员介绍给当地的学术和工业研究界。细胞使用区隔化来创建空间组织，使它们能够在不同的微环境中进行生化过程并控制生物分子结构。这个合作项目将测试细菌利用细胞内相分离来实现区隔化的假设，允许分子与细胞质快速交换，而不需要细胞膜。在压力下，细菌染色体被Dps蛋白重组成具有液晶性质的紧密凝聚物。为了确定Dps-DNA凝聚物的生物物理机制，单分子荧光显微镜将绘制凝聚物系统的相图，作为生理相关环境条件的函数。这些实验将补充活性粒子和连续体模型，这些模型将预测相分离形态和冷凝物的液态结晶度。通过主动微流变学测量凝析液的粘弹性和力学结构功能特性；偏振光显微镜可以识别出冷凝物中任何大的有序域。数学方法将确定结构和方向顺序，允许构建相分离和凝析物相顺序的微观模型。为了验证小分子可以在Dps:DNA凝聚中快速扩散以促进包括转录在内的酶活性的假设，我们将使用全内反射荧光显微镜直接测量凝聚液滴内分子的扩散。粒子轨迹的数学检验将揭示不同DNA区域的可达性，定量表征不同类型生物分子的运动性，促进对生物分子凝聚物的结构功能特性如何调节细胞活动的理解。该奖项由分子和细胞生物科学部（MCB）通过系统和合成生物学以及遗传机制项目，以及MPS数学科学部（DMS）通过数学生物学项目共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.