VARIATIONS IN DYNAMIC 3D STRUCTURE OF THE GOLGI

高尔基复合体是参与转运、翻译后修饰和分子分选的分泌途径的中心细胞器。分子进出高尔基体的机制是激烈争论的话题。需要回答三个问题。根据蓄水池成熟假说，分子是通过蓄水池内的高尔基体移动，还是通过小泡运输假说，通过小泡移动？第二，货物是通过囊泡、小管或两者同时沿顺行和逆行方向离开高尔基体？或者，所有机制可能一起起作用，这取决于碳运输的数量和细胞类型，池成熟可能比囊泡运输占优势。最后，我们计划确定反式高尔基体的组成、调节和内体/溶酶体途径的出口位点。基于我们早期的工作，我们的假设是，这些通道的出口来自不同的高尔基池。这些都是我们将在拟议的研究中解决的问题和假设。我们的方法是首先利用快速冷冻技术保存的细胞和组织，在高分辨率（5-7nm）下解决高尔基带的基本三维结构。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将使用只有组成分泌途径的NRK细胞，并将重点放在通过高尔基体的交通水平的调节上。胰脏的b细胞将在S.A. 2中使用，重点关注受调节的分泌途径。最后，来自哺乳期和非哺乳期啮齿类动物的乳腺上皮细胞将在sa 3中被专门用于研究一种被认为是池成熟模型的细胞。对每个池的表面积、体积、芽和小管的数量进行统计评估，将为评估哺乳动物高尔基体转运机制提供可靠的数据。

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.