Crowding and Confinement: Coupling of Bulk and Membrane Phase Separation in Giant Vesicles

一个活细胞的内部很小，只有十亿分之几立方厘米。它也非常拥挤。细胞内部包裹着数量惊人的不同分子——蛋白质、糖和核酸——是一个繁忙的空间。这个领域的许多分子参与者都是大型聚合物，有些不喜欢其他聚合物，有些更喜欢特定的邻居。因此，细胞空间不是无特征的流体。相反，它是高度纹理的——一种不同成分的共存液体的动态马赛克图案，就像熔岩灯中的油一样。这种分子行为统称为液-液相分离，在细胞内容物的组织和细胞功能的完成中起着重要作用。利用简化的细胞模型，最小化地模拟活细胞的大小和环境，目前的研究将研究这些分子分布如何在细胞样环境中组织起来，以及它如何影响最小细胞本身的边界和形状。该研究将由研究生和本科生研究人员在加州州立大学萨克拉门托分校（CSUS，主要是本科生和少数民族服务机构）和加州大学戴维斯分校之间的紧密研究合作伙伴关系中进行。技术概述：本提案旨在开发膜结合隔室的实验模型，以重现大分子拥挤环境中的液-液相分离。它研究了两种主要机制：(1)水溶性聚合物形成水两相体系的分离相和(2)凝聚生物聚合物的结合相分离。通过原位渗透诱导液-液分离，该研究验证了囊泡内大分子拥挤的变化是由内向外传递到膜边界激活膜界面的假设。激活包括分子（和结构域水平）重组和介观的全局形状转换。计划的活动包括三个主要的具体目标：(1)量化在囊泡约束下液-液分离过程中相粗化的动力学；(2)绘制渗透诱导的囊内大分子拥挤与膜重塑的关系；(3)表征包括重组生物聚合物在内的复杂囊泡内介质中液-液分离动力学与膜边界的耦合。实验方法结合湿化学和生化方法与荧光显微镜为基础的定量应用技术和定量图像分析。这项研究代表了加州大学戴维斯分校的研究生和加州州立大学萨克拉门托分校（CSUS，主要是本科生和少数民族服务机构）本科生之间的紧密合作关系。此外，这些研究活动将被用于加强加州大学戴维斯分校物理生物学的多部门课程，并将被纳入加州州立大学生物物理学的新本科课程。拟议的活动还将用于通过垂直整合计划（vertical - integrated - program）吸引代表性不足的STEM本科生，从而加强外展活动，该计划在本科教育期间提供多年的团队研究沉浸感。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。

NON-TECHNICAL SUMMARYThe inside of a living cell, a few billionths of a cubic centimeter, is tiny. It is also extremely crowded. Packing a staggeringly large number of different molecules – proteins, sugars, and nucleic acids – the cellular interior is a busy space. Many of the molecular actors in this space are large polymers with some disliking others, and others preferring specific neighbors. As a result, the cellular space is not a featureless fluid. Instead, it is highly textured – a dynamic mosaic pattern of co-existing liquids of different compositions like oil in lava lamps. Collectively referred to as liquid-liquid phase separation, this molecular behavior plays important roles in how cellular contents get organized and cellular duties accomplished. Using simplified models of cell, which minimally mimic the size and the environments of the living cell, present research will study how these molecular distributions in cell-like environment gets organized and how it affects the very boundary and the shape of the minimal cell itself. The research will be carried out by graduate students and undergraduate researchers in a tight research partnership between the California State University, Sacramento (CSUS, a primarily undergraduate and minority-serving institution) and the University of California, Davis. TECHNICAL SUMMARYThis proposal seeks to develop experimental models of membrane-bound compartments that recapitulate liquid-liquid phase separation in a macromolecularly crowded environment. It studies two major mechanisms: (1) segregative phase separation of water-soluble polymers producing aqueous two-phase systems and (2) associative phase separation of coacervating biopolymers. By osmotically inducing liquid-liquid phase separation in-situ, the effort tests the hypothesis that the changes in the intravesicular macromolecular crowding are transduced inside-out to the membrane boundary activating the membrane interface. The activation involves both molecular (and domain-level) reorganization and mesoscopic, global shape transformations. The planned activities include three major specific aims: (1) quantify the dynamics of phase coarsening during liquid-liquid phase separations in vesicular confinement; (2) map relations between osmotically-induced intravesicular macromolecular crowding and membrane remodeling; and (3) characterize the coupling of dynamics of liquid-liquid phase separation and membrane boundary in complex intravesicular media including reconstituted biopolymers. Experimental approaches combine wet chemical and biochemical methods with quantitative applications of fluorescence microscopy-based techniques and quantitative image analyses. The research represents a tight partnership between graduate students at UC Davis and undergraduate students California State University, Sacramento (CSUS, a primarily undergraduate and minority-serving institution). Additionally, the research activities will be leveraged to enhance a multi-department course in physical biology at UC Davis, and will be incorporated into a new biophysics undergraduate course at CSUS. The proposed activities will also be used to enhance outreach activities by engaging underrepresented undergraduates in STEM through the Vertically-Integrated-Program, which provides multi-year immersion in team-based research during their undergraduate education.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.