POWRE: Towards Functional Model Cells: Incorporating Internal Structure

这项工作的目标是合成内部复杂性接近其生物同行的仿细胞组件。尽管来自许多学科的科学家试图创造活细胞的合成复制品，但在体外只模拟了最简单的细胞结构和功能。到目前为止，大多数模型细胞都是脂质体。这些可能是质膜的优秀模型，但它们缺乏细胞内的组织。活细胞表现出内部有序性，根据有无将结构与胞浆分开的周围膜的存在，可将其分为两大类。POWRE奖支持的探索性工作旨在生成实验系统，在其中对这些类别的细胞内组织的每一种进行建模。创造具有较小内部囊泡的巨大囊泡并不难--事实上，当试图合成大的单层囊泡时，很难不做到这一点。这项工作将超越以往的研究，通过设计组成不同于模型质膜(外部囊泡)的模型细胞器(内部囊泡)。这将是朝着接近活细胞中发现的复杂程度迈出的重要一步。在巨大单层囊泡膨胀的过程中，通过在水相中包含小的囊泡，内部囊泡将被合并到巨大的单层囊泡(GUV)中。或者，预先形成的囊泡将通过显微注射被包裹在大脂质体中。“细胞器”的封装将通过视频增强光学显微镜进行验证(在某些情况下还会进行验证)。分子在“细胞器”和外部“细胞”之间的转移将通过荧光显微镜和流式细胞术进行。第二种模拟细胞内部结构的方法是通过大分子拥挤。大分子拥挤被认为是通过相分离来控制细胞内成分的结合，由于体积排斥，当存在高浓度的非相互作用的大分子时，相分离更容易发生。将首先用胶体颗粒(如乳胶微球和金属纳米棒或病毒颗粒)，然后用生物大分子(如白蛋白和微管蛋白或肌动蛋白)来研究“拥挤”对单个GUV含量的影响。高浓度的体积排除剂将被包裹在GUV内，以产生静态拥挤条件，并引发各向异性分子或颗粒的相分离。为了控制实验期间的“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由定量偏振光显微镜来跟踪。将通过监测双折射的变化(增加)来跟踪相分离成有序相的过程。将使用的其他观察内部有序性的方法包括反射光学显微镜(对于200 nm金属纳米棒)、荧光共振能量转移(FRET)和透射电子显微镜(TEM)。这项POWRE奖将允许基廷博士进行初步研究，以确定这些方法的可行性，并开始进行观察。该项目的长期发展如果完全成功，将导致科学家对细胞模型的看法发生变化，并将极大地促进对活细胞中分子自组装的理解，并将使这项研究成为基廷博士的一条独立的研究路线。

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.