POWRE: Towards Functional Model Cells: Incorporating Internal Structure

POWRE：走向功能模型细胞：合并内部结构

• 批准号: 0074845
• 负责人: Christine Keating
• 金额: $ 7.5万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0074845&HistoricalAwards=false
• 关键词: POWRE; Towards; Functional; Model; Cells

The goal of this work is to synthesize cytomimetic assemblies with internal complexity approaching that of their biological counterparts. Although scientists from many disciplines have attempted to create synthetic replicas of living cells, only the very simplest of cell structures and functions have been mimicked in vitro. To date, most model cells have been liposomes. These can be excellent models of the plasma membrane, but they lack intracellular organization. Living cells exhibit internal ordering which can be divided into two broad categories based on the presence or absence of a surrounding membrane separating the structure from the cytosol. The exploratory work supported by this POWRE award is aimed at generating experimental systems in which each of these classes of intracellular organization is modeled. It is not difficult to create giant vesicles having smaller internal vesicles - in fact, it can be hard not to, when attempting to synthesize large unilamellar vesicles. This work will move beyond previous studies by designing model organelles (inner vesicles) which differ in composition from the model plasma membrane (outer vesicle). This will be a significant step towards approximating the level of sophistication found in living cells. Internal vesicles will be incorporated within giant unilamellar vesicles (GUVs) by including small vesicles in the aqueous phase during swelling of giant unilamellar vesicles. Alternately, preformed vesicles will be encapsulated within large liposomes by microinjection. Encapsulation of "organelles" will be verified (and in some cases followed) by video-enhanced optical microscopy. Transfer of molecules between "organelles" and the outer "cell" will be followed by fluorescence microscopy and flow cytometry. A second approach to model the internal structure of cells is via macromolecular crowding. Macromolecular crowding has been postulated to control the association of intracellular components through phase segregation, which occurs more readily in the presence of high concentrations of noninteracting macromolecules due to volume exclusion. The effects of "crowding" on the contents of single GUVs will be investigated first with colloidal particles (e.g. latex microspheres and metal nanorods or virus particles), and then with biological macromolecules (e.g. albumin and tubulin or actin). High concentrations of volume excluders will be encapsulated within GUVs to generate static crowding conditions and initiate phase segregation of anisotropic molecules or particles. To control the "crowding" pressure during an experiment, GUV volume will be altered, e.g. via control of external osmolarity. Organization of internal particles and macromolecules will be followed primarily by quantitative polarized light microscopy. The process of phase-separation into ordered phases will be tracked by monitoring changes (increases) in birefringence. Other methods for observation of internal ordering that will be used include reflected light microscopy (for 200 nm metallic nanorods), fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). This POWRE award will allow Dr. Keating to perform preliminary studies to establish the feasibility of these approaches and to begin to make observations. The longer term development of this project will, if fully successful, lead to a change in the way scientists think about cell models, and will greatly advance understanding of molecular self-assembly in living cells, and will establish this research as an independent line of investigation for Dr. Keating.

这项工作的目的是将内部复杂性的细胞拟合组件综合，以接近其生物学对应物。 尽管来自许多学科的科学家试图创建活细胞的合成复制品，但只有最简单的细胞结构和功能才在体外模仿。迄今为止，大多数模型细胞一直是脂质体。这些可能是质膜的出色模型，但它们缺乏细胞内组织。活细胞表现出内部有序，可以根据周围膜与细胞质分开的膜分开的存在或不存在分为两种类别。该POWRE奖支持的探索性工作旨在生成实验系统，其中这些类别的细胞内组织都进行了建模。 创建具有较小内部囊泡的巨型囊泡并不难 - 实际上，在尝试合成大型Unilamellar囊泡时可能很难不。这项工作将通过设计模型细胞器（内部囊泡）与模型质膜（外部囊泡）不同，将超越先前的研究。这将是迈向近似活细胞中成熟水平的重要一步。 内部囊泡将通过在巨大的单层囊泡肿胀期间在水相中包含小囊泡来纳入巨型单层囊泡（GUV）。另外，预先形成的囊泡将通过显微注射封装在大型脂质体中。 “细胞器”的封装将被验证（在某些情况下），然后进行视频增强光学显微镜。分子在“细胞器”和外部“细胞”之间的转移将进行荧光显微镜和流式细胞仪。 对细胞内部结构进行建模的第二种方法是通过大分子拥挤。已经假定大分子人群通过相分离来控制细胞内成分的关联，由于体积排除量，在高浓度的非互动大分子的情况下，这种分离更容易发生。首先，将使用胶体颗粒（例如乳胶微球和金属纳米棒或病毒颗粒）研究“拥挤”对单个GUV内容的影响，然后再研究生物大分子（例如白蛋白和小白蛋白和小管蛋白或肌动蛋白）。高浓度的排除器将被封装在GUV中，以产生静态拥挤条件并启动各向异性分子或颗粒的相分离。为了控制实验中的“拥挤”压力，将改变GUV体积，例如通过控制外渗透压。内部颗粒和大分子的组织将主要进行定量极化光学显微镜。相位分离为有序阶段的过程将通过监视双折射的变化（增加）来跟踪。 观察内部排序的其他方法包括反射光学显微镜（对于200 nm金属纳米棒），荧光共振能量传递（FRET）和透射电子显微镜（TEM）。该POWRE奖将使Keating博士能够进行初步研究，以确定这些方法的可行性并开始进行观察。 如果完全成功，该项目的长期发展将导致科学家对细胞模型的思考方式发生变化，并大大提高人们对活细胞中分子自组装的理解，并将这项研究确立为基廷博士的独立研究。