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

拥挤和限制：巨囊泡中体相分离和膜相分离的耦合

• 批准号: 2342436
• 负责人: Atul Parikh
• 金额: $ 60万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2342436&HistoricalAwards=false
• 关键词: Crowding; Confinement; Coupling; Bulk; Membrane

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.

非技术总结活细胞内部（几亿分立厘米）很小。它也非常拥挤。细胞内部包装大量大量的不同分子（蛋白质，糖和核酸）是一个繁忙的空间。这个空间中的许多分子参与者都是大型聚合物，有些不喜欢其他分子，而另一些则喜欢特定的邻居。结果，蜂窝空间不是无特征的流体。取而代之的是，它具有高度纹理 - 一种动态的镶嵌模式，该模式是在熔岩灯中的油中共存液体（例如油中）的液体。这种分子行为统称为液态液相分离，在细胞含量的组织方式和完成细胞关闭方面起着重要作用。利用细胞的简单模型，这些模型最小化了活细胞的大小和环境，目前的研究将研究这些分子在类似细胞的环境中如何组织起来，以及它如何影响最小细胞本身的边界和形状。这项研究将由加利福尼亚州立大学（CSUS，小学本科和少数派服务机构）和加利福尼亚大学戴维斯分校之间的紧密研究合作伙伴关系，研究生和本科研究人员进行。技术摘要此提案旨在开发膜结合隔室的实验模型，以在大分子拥挤的环境中概括液态液相分离。它研究了两种主要机制：（1）产生水溶性聚合物的分离相分离，产生水溶性的两相系统和（2）（2）凝聚生物聚合物的缔合相分离。通过渗透诱导的液态液相分离，努力检验了以下假设：内部插入式大分子分子拥挤的变化被内部翻译成激活膜界面的膜边界。激活涉及分子（和域级）的重组和介观，全球形状转化。计划的活动包括三个主要的特定目的：（1）量化囊泡限制中液态液相分离期间变形的动力学； （2）渗透诱导的内部大分子拥挤与膜重塑之间的地图关系； （3）表征复杂的室内培养基（包括重构的生物聚合物）中液态相分离和膜边界动力学的耦合。实验方法将湿化学和生化方法与基于荧光显微镜的技术和定量图像分析的定量应用相结合。这项研究代表了加州大学戴维斯分校的研究生与萨克拉曼多加州州立大学的本科生之间的紧密合作伙伴关系（CSUS，小学和少数民族服务机构）。此外，将利用研究活动来增强加州大学戴维斯分校的物理生物学多部门课程，并将纳入CSUS的新生物物理本科课程中。 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 precious of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.