VARIATIONS IN DYNAMIC 3D STRUCTURE OF THE GOLGI

高尔基复合体是分泌途径的中心细胞器，参与转运、翻译后修饰和将设计到高尔基体后位置的分子分选。分子移动到高尔基体，通过高尔基体和从高尔基体出来的机制是激烈争论的话题。需要回答三个问题。根据脑池成熟假说，分子是通过脑池内的高尔基体移动，还是根据囊泡运输假说，分子是通过小囊泡移动？第二，货物退出高尔基体的顺行和逆行方向通过囊泡，小管或两者兼而有之？或者，所有的机制可以一起发挥作用，这取决于碳水化合物运输的量和细胞类型，脑池成熟可能优于囊泡运输。最后，我们计划确定出口网站从反式高尔基体的组成，调节和内体/溶酶体途径。我们的假设，基于我们早期的工作，是出口到每个这些途径是从不同的高尔基体池。这些都是我们将在拟议的研究中解决的问题和假设。我们的方法是首先使用快速冷冻技术最佳保存的细胞和组织，以高分辨率（5- 7 nm）解决高尔基带的基本3-D结构。所有这三个问题都将在我们的三个具体目标中得到解决。S.A. 1将使用NRK细胞，它只有一个组成性分泌途径，并将重点放在通过高尔基体的交通水平的调制。胰腺的B细胞将用于S.A. 2、关注调节分泌途径。最后，来自非泌乳和泌乳啮齿动物的乳腺上皮细胞将用于S.A.。3专门关注被认为是脑池成熟模型的细胞。统计评估表面积，体积和芽和小管从每个池延伸的数量将提供可靠的数据，以评估通过哺乳动物高尔基复合体的运输机制。

The Golgi complex is the central organelle of the secretory pathway involved in transport, post-translational modification and sorting of molecules designed to post-Golgi locations. The mechanism by which molecules move to, through and from the Golgi is the topic of intensive debate. Answers to three questions are required. Do molecules move through the Golgi within cisternae, according to the cisternal maturation hypothesis, or do they move via small vesicles, according to the vesicular transport hypothesis? Second, does cargo exit the Golgi in both the anterograde and retrograde direction via vesicles, tubules or both? Alternatively, all mechanisms may function together, depending on the amount of carbo trafficking and the cell type, cisternal maturation may predominate over vesicular traffic. Finally, we plan to identify the exit sites from the trans-Golgi for the constitutive, regulated and endosomal/lysosomal pathways. Our hypothesis, based on our earlier work, is that exit to each of these pathways is from different Golgi cisternae. These are the questions and hypotheses we will address in the proposed research. Our approach is to first solve the basic 3-D structure the Golgi ribbon at high resolution (5-7nm) using cells and tissues that are optimally preserved by rapid freezing technologies. All three questions will be addressed in each of our three specific aims. S.A. 1 will use NRK cells, which have only a constitutive secretory pathway, and will focus on modulation of the level of traffic through the Golgi. The b- cell of the pancreas will be used in S.A. 2, to focus on the regulated secretory pathway. Finally, the mammary epithelial cell from non- lactating and lactating rodents will be used in S.A. 3 to focus specifically on a cell that is considered a model of cisternal maturation. Statistical evaluation of surface area, volumes, and the number of buds and tubules extending from each cisterna will provide reliable data to evaluate the mechanism of transport through the mammalian Golgi complex.