Cages, Corrals and Vesicular Trafficking Shape the Diffusional Environment of the Plasma Membrane.

质膜是细胞内部与其环境之间的屏障。这些膜通常被描述为自由漂浮的脂质“海洋”，蛋白质“冰山”在“海洋”中自由扩散（移动）。在现实中，膜被分成小的区域，区域之间的运动受到膜嵌入结构的限制。这些限制扩散的结构将膜划分成具有特定功能的小区域，并且有多种类型的这些结构在不同的尺寸和时间尺度上控制扩散。我们对其中的两种结构——围栏和笼子感兴趣，目的是更好地理解这些结构的组成和动力学，并利用这些信息，建立一个蛋白质扩散模型，解释笼子和围栏的影响。栅栏是由附着在细胞“骨架”上的“尖桩”蛋白质形成的“尖桩栅栏”，并穿过膜形成“渗透屏障”，限制了从栅栏的流出。我们最近的工作确定，栅栏可以选择性地捕获旧蛋白质，然后将其内吞（内化）以将其从膜上移除。我的NSERC研究项目最近发现的笼子更为神秘。它们的组成和作用尚不清楚，但它们在较短的时间内就像畜栏一样。我们已经初步确定了蛋白质谱蛋白作为笼的结构成分，并开发了直接成像细胞膜中的笼和栅栏所需的工具。在目标1中，我们将识别和表征形成笼状结构的蛋白质。在目标2中，我们将讨论畜栏是否选择性地从质膜上去除旧的蛋白质，以及新蛋白质的释放（胞吐）是否清除畜栏周围的膜。最后，我们将继续开发细胞膜中蛋白质扩散的数学模型，因为目前的模型没有考虑笼子和畜栏的作用。我们已经证明，连续时间随机漫步模型最能描述畜栏和笼子对一种蛋白质扩散的影响；在目标3中，我们将使用一组代表膜蛋白主要结构类别的蛋白质来确定该模型是否为蛋白质扩散的一般模型。从长远来看，该项目将揭示用于在细胞表面组织蛋白质的过程，而在短期内，它将确定关键的结构机制及其组成。通过该项目培训的HQP获得了显微镜和计算机辅助分析方面的经验，以前的HQP利用这些专业知识开始了开发下一代MRI成像和组织学样本疾病诊断软件的职业生涯。这个项目将继续我在HQP高级显微镜和图像分析方面的成功培训，为这些学员提供显微镜和图像处理行业急需的技能。

The plasma membrane is the barrier between the inside of a cell and its environment. These membranes are often described as "seas" of free-floating lipids with protein "icebergs" that freely diffuse (move) through the "sea". In reality, membranes are divided into small regions, with movement between regions restricted by membrane-embedded structures. These diffusion-restricting structures divide membranes into small regions that take on specialized functions, and there are multiple types of these structures that control diffusion over different size and time scales. We are interested in two of these structures - corrals and cages, with the goal of better understanding the composition and dynamics of these structures, and using this information, develop a model of protein diffusion that accounts for the effects of cages and corrals. Corrals are "picket-fences" created by "picket" proteins that are attached to the cells "skeleton", and extend through the membrane to create a "percolation barrier" that limits egress from the corral. Our recent work determined that corrals may selectively trap old proteins, which are then endocytosed (internalized) to remove them from the membrane. Cages - recently discovered by my NSERC research program - are more mysterious. Their composition and role are unknown, but they act like corrals over shorter periods of time. We have tentatively identified the protein spectrin as a structural component of cages, and developed the tools needed to directly image cages and corrals in the cell membrane. In aim 1 we will identify and characterize the proteins which form cages. In aim 2 we will address whether corrals selectively remove older proteins from the plasma membrane, and whether the release (exocytosis) of new proteins clears the surrounding membrane of corrals. Lastly, we will continue our development of a mathematical model of protein diffusion in the cell membrane, as current models do not account for the role of cages and corrals. We have shown that the Continuous Time Random Walk model best describes the effect of corrals and cages on the diffusion of one protein; in aim 3 we will use a panel of proteins representing the major structure classes of membrane proteins to determine whether this model is a general model of protein diffusion. In the long-term this program will uncover the processes used to organize proteins on the cell surface, and in the short-term it will identify the key structuring mechanisms and their composition. The HQP trained by this program gain experience in microscopy and computer-aided analyses, with previous HQP using this expertise to launch careers developing the next generation of software for diagnosing diseases in MRI imaging and in histological samples. This program will continue my successful training of HQP in advanced forms of microscopy and image analysis, providing these trainees with skills which are highly in-demand in the microscopy and image processing industries.