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

技术描述：这一建议是基于这样一种想法，即即使在没有特定的生物分子相互作用的情况下，也有可能实现亚细胞组织的关键结构和功能方面，这是因为在充满大分子的溶液中出现的物理化学现象，即使没有特定的生物分子相互作用。特别令人感兴趣的是水相分离，这通常发生在聚合物溶液中，并提供了一种隔间的方法。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是非膜细胞器的存在和性质的主要原因，(2)这些结构反过来可以作为膜组装的模板。基于RNA、多胺、蛋白质和中性聚合物的拥挤溶液，将开发亚细胞胞质和核隔室的实验模型。蛋白质磷酸化将被用来驱动微室的形成和拆卸，这些微室将作为模型细胞器。提出了三个研究目标：(1)基于响应于蛋白质磷酸化状态而形成的水相室的模型非膜细胞器的动态组装/拆卸。(2)模型细胞器周围形成模板膜。(3)细胞大小的脂泡中的有丝分裂/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，膜结合的细胞器(如细胞核)和非膜性细胞器(如核仁、P颗粒)都发挥着关键的细胞功能。推动这些研究的中心问题是：相对非特定的化学和物理效应在生物细胞的亚细胞组织和相关功能中扮演什么角色，以及少量简单的分子成分是如何产生复杂性的？研究人员假设，尽管所提出的模型细胞故意简单，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和富含蛋白质的隔室的可逆形成和溶解，围绕先前存在的富含蛋白质的隔间形成内膜，最后在非常原始的有丝分裂细胞的模拟中模拟细胞分裂。更广泛的影响。两名研究生将在分子和细胞生物学、化学、生物物理学和材料科学的界面上接受培训。本科生将在课程学分的学年和暑期通过各种现场REU计划招募本科生参与该项目，目标是在学年招收一名学生，在暑期招收一到两名学生。预算中包括了每个暑假一名K-12教师的支助。调查人员将与PSU MRSEC中现有的教师研究经验计划合作。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教室。真实世界的例子超越了激发这项提议的智力价值的细胞内细胞器，还包括例如食品科学、药物输送和环境补救。该奖项由MCB/BIO的系统和合成生物学集群资助，并由CHE/MPS的生命过程化学计划共同资助。

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.