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Experimental Model Systems for Intracellular Compartmentalization: Dynamic Formation/Disassembly of Model Organelles in Artificial Cells

细胞内区室化的实验模型系统：人工细胞中模型细胞器的动态形成/分解

基本信息

批准号：
1244180
负责人：
Christine Keating
金额：
$ 81.25万
依托单位：
Pennsylvania State Univ University Park
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2013
资助国家：
美国
起止时间：
2013-03-01 至 2018-02-28
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244180&HistoricalAwards=false
关键词：
Experimental Model Systems Intracellular Compartmentalization

项目摘要

Technical Description: This proposal is based on the idea that it may be possible to realize key structural and functional aspects of subcellular organization due to the physicochemical phenomena that arise in solutions that are crowded with macromolecules "like the cytoplasm and nucleoplasm are" even in the absence of specific biomolecular interactions. Of particular interest is aqueous phase separation, which commonly occurs in polymer solutions and offers a means of compartmentalization. This work will evaluate two hypotheses: (1) that phase separation in the crowded intracellular environment could be largely responsible for the existence and properties of non-membranous organelles, and (2) that these structures could in turn serve as templates for membrane assembly. Experimental models of subcellular cytoplasmic and nuclear compartments will be developed based on crowded solutions of RNA, polyamines, proteins, and neutral polymers. Protein phosphorylation will be used to drive formation and disassembly of microcompartments that will serve as model organelles. Three research objectives are proposed: (1) Dynamic assembly/disassembly of model non-membranous organelles based on aqueous phase compartments that form in response to protein phosphorylation state. (2) Templated membrane formation around these model organelles. (3) Primitive model for mitosis/cell cycle in cell-sized lipid vesicles that contain the crowded solution and phase compartment-based model organelles. Nontechnical Description: Intracellular organization is a hallmark of living cells, with both membrane-bounded (e.g., nucleus) and non-membranous organelles (e.g., nucleolus, P-granules) performing key cellular functions. The central questions driving these investigations are: What role do relatively nonspecific chemical and physical effects play in subcellular organization and the associated functions of biological cells, and how does complexity arise from a small number of simple molecular components? The investigators hypothesize that, despite the deliberate simplicity of the proposed model cells, they will be able to mimic complex biological processes and specific biochemical interactions, such as the reversible formation and dissolution of RNA and protein-rich compartments, the formation of interior membranes around pre-existing protein-rich compartments, and finally to model cell division in a very primitive mimic of the mitotic cell. Broader Impacts. Two graduate students will be trained at the interface of molecular and cell biology, chemistry, biophysics and materials science. Undergraduate students will be recruited to work on this project during the academic year for course credit and in the summer through the various on-site REU programs, with a target of one student during the academic year and one or two students during the summer. Support for one K-12 teacher each summer is included in the budget. The investigators will team with the existing Research Experiences for Teachers program in the PSU MRSEC. Teachers will perform experiments and develop curriculum materials to bring back to their own classrooms the following school year. Real-world examples go beyond the intracellular organelles that motivate the intellectual merit of this proposal to also include, e.g., food science, drug delivery, and environmental remediation.This award is being funded by the Systems and Synthetic Biology Cluster in MCB/BIO and co-funded by the Chemistry of Life Processes Program in CHE/MPS.

技术描述：该提案基于这样的想法：即使在没有特定的生物分子相互作用的情况下，由于在充满大分子“如细胞质和核质”的溶液中出现的物理化学现象，也有可能实现亚细胞组织的关键结构和功能方面。特别令人感兴趣的是水相分离，它通常发生在聚合物溶液中并提供了一种分隔方式。这项工作将评估两个假设：（1）拥挤的细胞内环境中的相分离可能在很大程度上负责非膜细胞器的存在和特性，（2）这些结构反过来可以充当膜组装的模板。亚细胞细胞质和核区室的实验模型将基于 RNA、多胺、蛋白质和中性聚合物的拥挤溶液来开发。蛋白质磷酸化将用于驱动作为模型细胞器的微区室的形成和分解。提出了三个研究目标：（1）基于响应蛋白质磷酸化状态形成的水相区室的模型非膜细胞器的动态组装/拆卸。 (2)这些模型细胞器周围的模板化膜形成。 (3) 细胞大小的脂质囊泡中的有丝分裂/细胞周期的原始模型，其中包含拥挤的溶液和基于相室的模型细胞器。非技术描述：细胞内组织是活细胞的标志，膜界细胞器（例如细胞核）和非膜细胞器（例如核仁、P 颗粒）执行关键的细胞功能。推动这些研究的核心问题是：相对非特异性的化学和物理效应在亚细胞组织和生物细胞的相关功能中发挥什么作用，以及少量简单的分子成分如何产生复杂性？研究人员推测，尽管所提出的模型细胞故意简单化，但它们将能够模拟复杂的生物过程和特定的生化相互作用，例如RNA和富含蛋白质的区室的可逆形成和溶解，在预先存在的富含蛋白质的区室周围形成内膜，最后在有丝分裂细胞的非常原始的模拟中模拟细胞分裂。更广泛的影响。两名研究生将接受分子和细胞生物学、化学、生物物理学和材料科学交叉领域的培训。将在学年期间招募本科生参与该项目，以获得课程学分，并在夏季通过各种现场 REU 项目招募本科生，目标是在学年期间招收一名学生，在夏季招收一到两名学生。每年夏天对一名 K-12 教师的支持已包含在预算中。研究人员将与 PSU MRSEC 现有的教师研究经验项目合作。教师将进行实验并开发课程材料，以便在下一学年带回自己的课堂。现实世界的例子超出了激发该提案智力价值的细胞内细胞器，还包括食品科学、药物输送和环境修复等。该奖项由 MCB/BIO 系统和合成生物学集群资助，并由 CHE/MPS 生命过程化学项目共同资助。