Dynamics of Secretory Membrane Trafficking, Sorting and Compartmentalization

Child Health And Human Development

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Abstract

We have used fluorescent protein technology to investigate the characteristics of endomembrane organization in eukaryotic cells, including polarized cell monolayers in tissue culture and in living embryos. Our research has focused on the subcellular localization, mobility, transport routes and binding interactions of proteins with regulatory roles in the organization of membrane traffic and compartmentalization. [unreadable] [unreadable] In one project we have studied the biogenesis and inheritance of the Golgi apparatus. Golgi inheritance during mammalian cell division is known to occur through the disassembly, partitioning, and reassembly of Golgi membranes but the mechanisms responsible for these processes are poorly understood. To address these mechanisms, we examined the identity and behavior of Golgi proteins within mitotic membranes using dynamic cell imaging of Golgi and ER markers, electron microscopy, ER fragmentation with ionomycin, and ER entrapment through misfolding. Two overall conclusions were drawn from the data. First, that mitotic Golgi haze, seen in metaphase, represents recycled Golgi proteins trapped in the ER, a consequence that is likely related to the mitosis-specific disassembly of ER exit sites and inactivation of Arf1. Second, that mitotic Golgi fragments, seen in prometaphase and telophase, are not isolated breakdown products of the Golgi; rather, they are structures undergoing continuous exchange of their components through the ER and dispersed ER exit sites. These conclusions suggest a model in which the Golgi is inherited through the ER in mitosis and that mitotic Golgi disassembly/reassembly involves the inhibition and subsequent reactivation of cellular activities that control recycling of Golgi components into and out of the ER. Evidence supporting the first of these conclusions- that Golgi haze in metaphase cells represents Golgi proteins within the ER, came from three lines of evidence: 1) mitotic haze can be resolved into ER by high-resolution confocal microscopy, 2) it redistributes with ER into fragments upon ionomycin treatment, and 3) it displays quality control features characteristic of ER such as misfolding and retention of proteins. Evidence that mitotic Golgi fragments observed in prophase and telophase represent ER-derived structures through which Golgi proteins cycle rapidly (i.e., from ER exit sites to a fragment and then back again into the ER) came from fluorescence double-labeling, immunoelectron microscopy, and photobleaching recovery experiments. Live cell imaging of a single cell co-expressing Sec13-YFP and GalT-CFP revealed that mitotic Golgi fragments grow out from ER export domains at the end of mitosis, remain near these sites for a short period, and then undergo clustering into a Golgi ribbon. Immunoperoxidase electron microscopy of cells in prometaphase and telophase further showed that mitotic Golgi fragments were clusters of tubules/vesicles localized adjacent to ER export sites, and in some cases, were in direct continuity with ER export domains. Finally, when a mitotic Golgi fragment was photobleached in cells expressing GalT-YFP, fragment fluorescence rapidly recovered most of its original intensity within 2 min, indicating Golgi proteins continuously move in and out of mitotic fragments while maintaining steady-state pools in these fragments.[unreadable] In a second project we used live cell imaging approaches to investigate the organization of the secretory pathway in the fly embryo. We specifically asked how this pathway and its organelles become equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. We found that the endoplasmic reticulum and Golgi apparatus became segregated among nuclei only when these nuclei migrated to the periphery of the embryo at nuclear division cycle 10. The nuclear-associated units of ER and Golgi across the syncytial blastoderom produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalize around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.