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.

非技术性总结活细胞的内部只有十亿分之一立方厘米，非常微小。 它也非常拥挤。细胞内部是一个忙碌的空间，它容纳了大量不同的分子--蛋白质、糖和核酸。这个空间中的许多分子演员都是大聚合物，有些人不喜欢其他人，而其他人则喜欢特定的邻居。 因此，细胞空间不是一种无特征的流体。 相反，它是高度纹理-一个动态的马赛克图案共存的液体不同的成分，如石油在熔岩灯。 这种分子行为统称为液-液相分离，在细胞内容物如何组织和细胞职责如何完成方面起着重要作用。 使用简化的细胞模型，最小限度地模仿活细胞的大小和环境，本研究将研究这些分子分布在细胞样环境中是如何组织的，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伙伴关系中进行。 技术概述该建议旨在开发膜结合隔室的实验模型，其重现了在大分子拥挤环境中的液-液相分离。它研究了两种主要的机理：（1）水溶性聚合物的分离相分离，产生含水两相系统和（2）凝聚生物聚合物的缔合相分离。通过原位诱导液-液相分离，该努力测试了这样的假设，即囊内大分子拥挤的变化由内向外转导到膜边界，从而激活膜界面。激活涉及分子（和域水平）重组和介观，全球形状转换。计划的活动包括三个主要的具体目标：（1）量化在囊泡限制液-液相分离过程中的相粗化动力学;（2）绘制由药物诱导的囊泡内大分子拥挤和膜重塑之间的关系;（3）描述液-液相分离动力学和复杂囊泡内介质（包括重组生物聚合物）中膜边界的耦合。实验方法将联合收割机湿化学和生物化学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和加州州立大学，萨克拉门托（CSUS，一个主要为本科生和少数民族服务的机构）的本科生之间的紧密合作关系。 此外，研究活动将被用来加强加州大学戴维斯分校的物理生物学多部门课程，并将被纳入CSUS的新生物物理学本科课程。拟议的活动还将用于加强外展活动，通过垂直整合计划（Vertically-Integrated-Program）吸引在STEM领域代表性不足的本科生，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NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。