Dynamics of Secretory Membrane Trafficking, Sorting and

Child Health And Human Development

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Abstract

The Section on Organelle Biology investigates the global principles underlying secretory membrane trafficking, sorting and compartmentalization within eukaryotic cells. Live cell imaging of green fluorescent protein (GFP) fusion proteins in combination with photobleaching and photoactivation techniques are being used to investigate the subcellular localization, mobility, transport routes and binding interactions of a variety of proteins with important roles in the organization and regulation of membrane traffic and compartmentalization. Quantitative measurements of these protein characteristics are used in kinetic modeling and simulation experiments in order to test mechanistic hypotheses related to protein and organelle dynamics. Among the topics currently under study include: the mechanism of Golgi dissassembly and ressembly during mitosis; membrane binding/dissociation kinetics of cytosolic machinery involved in COPI coat formation and its role in protein transport through the secretory pathway; the basis for the origin and proliferation of peroxisomes; and the foundation for compartmentalization of endomembranes in the developing Drosophilia embryo. In addition to these topics, a major effort in the lab has been to develop new fluorescence microscopy techniques. Towards this end, we have helped develop a new method called photoactivated localization microscopy (PALM) for imaging fluorescently tagged proteins at near molecular resolution, and have introduced a technique called fluorescence protease protection (FPP) for determining the topology of fluorescently-tagged proteins in living cells.[unreadable] [unreadable] Development of green fluorescent protein technology[unreadable] Patterson, Sougrat, Lorenz, and Hailey in collaboration with Betzig, Hess, Davidson and Bonifacino[unreadable] [unreadable] [unreadable] Over the past year, we have continued our focus on the development of new fluorescence imaging approaches. One area has been the development of a high-resolution microscopy technique capable of optical resolutions beyond the limit imposed by diffraction. This technique was developed in collaboration with Dr. Eric Betzig (Howard Hughes Medical Institute, Janelia Farm Research Campus and New Millennium Research), Dr. Harald Hess (HHMI, Janelia Farm and NuQuest Research) and members of the Juan Bonifacino lab (CBMB), and the Michael Davidson lab (Florida State University). Termed photoactivated localization microscopy (PALM), the method involves serial photoactivation and subsequent bleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The aggregate position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities (up to 105 molecules/?m2). PALM imaging of intracellular structures (including lysosome, Golgi apparatus and mitochondria) in cryo-prepared thin sections was demonstrated, as well as imaging of vinculin and actin in fixed cells with TIRF excitation, and correlative PALM/transmission electron microscopy of a mitochondrial marker protein. [unreadable] A second new fluorescent protein technique developed in our lab allows a protein?s topology to be determined in living cells. Termed fluorescence protease protection (FPP), the assay provides a fluorescent readout in response to trypsin-induced destruction of GFP attached to a protein-of-interest before and after plasma membrane permeabilization. In performing the FPP assay, a fluorescent protein is attached to the N or C terminus of a protein of interest. Subsequently, cells expressing the fusion protein are exposed to trypsin either before or after plasma membrane permeabilization by digitonin. If the fluourescent protein moiety on the expressed protein faces the environment exposed to trypsin (that is the cytoplasm), then its fluorescent signal will be lost. Conversely, if the fluorescent protein moiety on the expressed protein faces the environment protected from trypsin (that is, the lumen of a compartment) then its fluorescence persists. Given these outcomes and the fluorescent protein?s known engineered position within the protein, it is possible to deduce the orientation of the protein across the lipid bilayer. We demonstrated the broad applicability of FPP by using it to define the topology of proteins localized to several different organelles, including the ER, Golgi apparatus, mitochondria, peroxisomes and autophagosomes. [unreadable] [unreadable] Golgi biogenesis and inheritance [unreadable] [unreadable] 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.[unreadable] 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.