Dynamics and organization of the transitional endoplasmic reticulum and the Golgi apparatus

The Golgi apparatus performs central functions in the secretory pathway by processing and sorting secretory material and synthesizing certain lipids. These biochemical events are well characterized, but the mechanism by which secretory cargo flows through the Golgi is an area of intense debate. A popular model for Golgi organization is the maturation model, which proposes that Golgi cisternae are transient structures that progress through the stack, maturing in the process, until they ultimately break up into secretory and transport vesicles. In the maturation model, new Golgi cisternae are produced by the coalescence of ER-derived COPII vesicles. These vesicles bud from specialized subdomains of the ER called transitional ER (tER) sites. Thus, the tER represents the birthplace of the Golgi. To gain a better understanding of the Golgi, we need to determine the nature of the tER. The tER has not been well characterized in the past due to the technical difficulty of visualizing this compartment. I overcame this hurdle by developing two methods for examining the tER that have allowed me to greatly extend our understanding. First, I devised an improved immunofluorescence protocol that yields superb preservation of tER sites and other small cellular structures. Using this protocol, I was able to observe the organization of the tER in mammalian cells under a variety of conditions. I also used this protocol to quantify the relationship between tER activity and Golgi disassembly during mammalian mitosis. Second, I developed a system for accelerating the acquisition of 4D image sets of living cells. This Fast-4D system, in combination with a GFP-tagged tER marker allowed for a detailed analysis of tER sites in the yeast Pichia pastoris. From these experiments we concluded that the tER is an independent long-lived ER subdomain that is not directly positioned by the cytoskeleton. Our video data show that tER sites can form de novo and are capable of fusing with one another but do not appear to divide. These observations have led to a model that explains the organization and propagation of tER sites.