We investigate the global principles underlying secretory membrane trafficking, sorting, and compartmentalization within eukaryotic cells. We use live-cell imaging of green fluorescent protein (GFP) fusion proteins in combination with photobleaching and photoactivation techniques 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. Using quantitative measurements of these protein characteristics in kinetic modeling and simulation experiments, we test mechanistic hypotheses related to protein and organelle dynamics. The topics currently under study include growth and maintenance of endoplasmic reticulum (ER) and Golgi morphology in mammalian cells and developing Drosophilaembryos; the mechanism(s) of secretory protein transport into and out of the Golgi apparatus; membrane binding/dissociation kinetics of trafficking machinery and its regulation; the generation and maintenance of cell polarity; protein aggregation in the cytoplasm and nucleus; the biogenesis and maintenance of peroxisomes and autophagosomes; and Golgi breakdown and reassembly during mitosis.
Development of green fluorescent protein technology
Patterson, Lorenz, Hailey, Ward,1 Snapp2
Over the past year, we continued our efforts to optimize numerous live-cell imaging approaches, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation, in order to analyze the dynamics of fluorescently labeled proteins expressed in living cells. In addition, we recently developed a fluorescence protease protection (FPP) assay adapted from classic protease protection assays. It provides a fluorescence read-out upon trypsin-induced destruction of GFP attached to a protein of interest before and after plasma membrane permeabilization. We used the FPP technique to define the topology of transmembrane and lumenal proteins localized within diverse cellular compartments, including the ER, Golgi apparatus, peroxisomes, and mitochondria. In addition, we used the same technique to detect minor populations of fluorescently tagged proteins localized within membrane-bound structures such as autophagosomes, which are otherwise masked by the protein’s population in the cytoplasm.
Lippincott-Schwartz J. Dynamics of secretory membrane trafficking. Ann NY Acad Sci 2005;1038:1-10.
Lippincott-Schwartz J, Snapp E. Imaging of membrane systems and membrane traffic in living cells. In: Spector D, ed. Live Cell Imaging: A Laboratory Manual. Woodbury, NY: Cold Spring Harbor Press, 2004;140-165.
Ward T, Lippincott-Schwartz J. The uses of GFP in mammalian cells. In: Chalfie M, Kain S, eds. Green Fluorescent Protein: Properties, Applications and Protocols. New York: Wiley-Liss, 2004;105-125.
Lipid raft dynamics and cell polarity
Mavrakis, Polishchuk3; in collaboration with Arias, Hodge, Kenworthy4
The membrane microdomains enriched with cholesterol and glycosphingolipids known as lipid rafts serve as platforms for protein segregation and signaling. Despite extensive research, it is not clear whether raft domains are mobile structures, if protein associations with rafts are stable or transient, or how perturbations of raft structure affect the dynamics of individual proteins. To address these issues, we used FRAP to test whether raft association affects a protein’s ability to diffuse laterally over large distances across the cell surface. We systematically measured the diffusion coefficients (D) of several types of raft and nonraft proteins under steady-state conditions and in response to raft perturbations. We found that, under normal conditions, individual raft proteins diffused freely over large distances (more than 4 micrometers) and exhibited Ds that varied 10-fold, indicating that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membranes (including cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins, indicating that raft association is not the dominant factor in determining protein mobility at the cell surface and ruling out several models for raft dynamics, including stable immobile rafts and stable mobile rafts. Assuming that raft domains exist, our data imply that raft proteins must rapidly partition into and out of the domains.
It is thought that lipid rafts are important for sorting glycosylphosphatidylinositol (GPI)-anchored proteins to the apical plasma membrane of polarized cells, with GPI-anchored proteins segregating into raft-enriched carriers in the trans-Golgi network (TGN) and then trafficking directly to the apical plasma membrane. This view relies on the localization of cholesterol-enriched lipid rafts at both the TGN and apical plasma membrane and on the affinity of GPI-anchored proteins for rafts. To test this model in living cells, we studied the pathway for apical delivery of GPI-anchored proteins tagged with GFP in polarized MDCK cells. We employed confocal microscopy and treatment with tannic acid, a cell-impermeant fixative that inhibits plasma membrane fusion within seconds and does not pass through tight junctions. We found that the GPI-anchored proteins followed an indirect, transcytotic route rather than trafficking directly from the TGN to the apical plasma membrane, as previously thought. The proteins first exited the TGN in membrane-bound carriers that also contained basolateral cargo (such as the VSVG protein), although the two cargos were laterally segregated. The carriers were then targeted to and fused with a zone of lateral plasma membrane adjacent to tight junctions, a zone that is known to contain the exocyst. Thereafter, the GPI-anchored proteins, but not basolateral cargo, were rapidly internalized, together with endocytic tracer, into clathrin-free transport intermediates that trans-cytosed to the apical plasma membrane. The data revealed that apical sorting of GPI-anchored proteins occurs at the plasma membrane rather than at the TGN and involves apically directed transcytotic carriers derived from basolateral membranes.
Goodwin JS, Drake KR, Rogers C, Wright L, Lippincott-Schwartz J, Philips MR, Kenworthy AK. Depalmitoylated Ras traffics to and from the Golgi complex via a non-vesicular pathway. J Cell Biol 2005;170:261-272.
Kenworthy AK, Nichols BJ, Remmert CL, Hendrix GM, Kumar M, Zimmerberg J, Lippincott-Schwartz J. Dynamics of lipid rafts at the cell surface. J Cell Biol2004;165:735-746.
Murray JL, Mavrakis M, McDonald NJ, Yilla M, Sheng J, Bellini WJ, Zhao L, Le Doux JM, Shaw MW, Luo CC, Lippincott-Schwartz J, Sanchez A, Rubin DH, Hodge TW. Rab9 GTPase is required for the replication of HIV-1, filoviruses, and measles virus. J Virology 2005;79:11742-11751.
Polishchuk R, Di Pentima A, Lippincott-Schwartz J. Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway. Nat Cell Biol 2004;6:297-307.
Wakabayashi Y, Lippincott-Schwartz J, Arias W. Intracellular trafficking of bile salt export pump (ABCB11) in polarized hepatic cells: constitutive cycling between the canalicular membrane and rab11-positive endosomes. Mol Biol Cell 2004;15:3485-3496.
Dynamics and differentiation of the endoplasmic reticulum, peroxisomes, and autophagosomes
Lorenz, Mavrakis, Hailey, Kim, Snapp,2 Frescas5; in collaboration with Brandizzi, De Lotto, Hegde, Lilly
The Drosophila embryo and ovary provide two unique model systems for studying changes in the organization and dynamics of the ER during development. To follow ER dynamics in the syncytial blastoderm, a multinucleated single-cell embryo, we expressed the GFP-tagged ER marker lysosome-KDEL in living Drosophila embryos and performed time-lapse confocal microscopy. We found that cortical ER exists as a single interconnected membrane system when nuclei are still localized in the embryo interior. How this organization of ER changes upon nuclei arrival at the embryo cortex and whether it is dependent on microtubules or actin are questions currently under investigation. The answers should help clarify how secretory membranes are equivalently partitioned among nuclei before enclosure of each nucleus by plasma membrane at cellularization. To study ER organization in the Drosophila ovary, we visualized, in collaboration with Mary Lilly, GFP-tagged reporters in ovarian cysts. We found that the ER contained fusomal membranes, which associate with cytoskeletal proteins and branch along spindle equators, physically connecting all cells within a cyst. We also found that ER proteins are capable of diffusing freely throughout the membranes, indicating that the ER exists as a single membrane system shared by all cystocytes in dividing ovarian cysts.
Many newly synthesized proteins that are misfolded or unassembled in the ER undergo retrograde translocation into the cytoplasm, where cytosolic proteases degrade them. To investigate the degradative pathway, we tagged photoactivatable (PA) GFP to ER-associated degradation (ERAD) substrates to visualize their fate in vivo. The photoactivatable GFP is ideal for such analysis in that only GFP molecules that have been photoactivated are visible within cells, allowing protein turnover to be measured in the absence of new protein synthesis. Using the PA-GFP–tagged ERAD substrates, we are studying the role of ubiquitin ligases and other regulatory molecules involved in ERAD. We are also studying the fate of ERAD substrates when proteasome activity is inhibited. Given that our data suggest that many ERAD substrates accumulate in autophagosomes when proteasome activity is inhibited, our work has led us to the area of autophagosome biogenesis.
Peroxisomes are small membrane-bound organelles present in virtually all eukaryotic cells that function in lipid metabolism and defense against oxidative stress. It is unclear, however, how peroxisomes are formed and maintained in mammalian cells. We are investigating the biogenesis of peroxisomes by studying the trafficking itinerary of PEX16, which acts upstream of PEX3 and PEX19 in the formation of peroxisomes in mammalian cells. We employed green fluorescent protein (mGFP) and photoactivatable mGFP-tagged versions of PEX16 in photobleaching and pulse-photolabeling experiments as well as in vitro binding assays. Our data suggest that PEX16 is co-translationally translocated into the ER membrane and then sorts to peroxisomes by a process involving peroxisome outgrowth from the ER. Our data suggest that peroxisomes are derived from and are maintained by the ER and are not autonomous organelles.
daSilva LLP, Snapp E, Denecke J, Lippincott-Schwartz J, Hawes C, Brandizzi F. ER exit sites and Golgi bodies in plant cells form mobile, secretory units. Plant Cell 2004;16:1753-1771.
Shen J, Snapp EL, Lippincott-Schwartz J, Prywes R. Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response. Mol Biol Cell 2005;25:921-932.
Snapp E, Iida T, Frescas D, Lippincott-Schwartz J, Lilly M. The fusome mediates intercellular ER connectivity in Drosophila ovarian cysts. Mol Biol Cell 2004;15:4512-4521.
Snapp E, Reinhart G, Bogert B, Lippincott-Schwartz J, Hegde R. The organization of engaged and quiescent translocons in the endoplasmic reticulum of mammalian cells. J Cell Biol 2004;164:997-1007.
Molecular basis for Golgi biogenesis and function
Altan-Bonnet, Liu, Patterson, Sougrat; in collaboration with Hirschberg, Jackson, Phair
The Golgi apparatus serves many functions that are essential for cell growth and homeostasis, including protein sorting, processing, and transport within the secretory pathway. In addition, the Golgi acts as a membrane scaffold to which diverse signaling, sorting, and cytoskeleton proteins adhere. To understand how newly synthesized proteins traffic through the Golgi apparatus, we developed a protocol that involves selective highlighting of GFP-tagged cargo proteins in the Golgi and monitoring of the distribution and export kinetics of the cargo molecules as they pass through the Golgi apparatus. Using this approach in combination with conventional biochemical and ultrastructural methods, we are seeking to obtain new information regarding the mechanism of transport of proteins through the Golgi apparatus. We aim to determine whether the numerous cisternae constituting the Golgi act as cargo carriers in a process of cisternal maturation or whether the cisternae are stable elements with cargo transferred between them by small vesicles or direct connections. We also are using the kinetic analysis tools of computational cell biology to test different models of cargo transport mediated by trafficking machinery. One model incorporates reversible partitioning of cargo into membrane domains enriched in either glycerophospholipids characteristic of the ER or glycosphingolipids characteristic of the plasma membrane.
Cytosolic coat proteins that bind reversibly to membranes carry out a central role in membrane trafficking by concentrating macromolecules into specialized membrane patches that deform into coated buds to produce coated carriers. The coatomer (COPI)-type coat helps mediate protein sorting and transport within the Golgi apparatus. Binding of COPI to membranes is regulated by the small GTPase Arf1, which, in its GTP-bound state, is active and assembles coats and, in its GDP-bound state, is inactive and disassembles coats. We used FRAP, FLIP, and confocal time-lapse imaging to investigate the in vivo dynamics of GFP-tagged versions of COPI, Arf1, ArfGAP1 (which catalyzes Arf1 GTP hydrolysis), and GBF1 (the Arf1 exchange factor that catalyzes GTP-for-GDP exchange on Arf1). The techniques allowed us to clarify the specific roles that the components play in the assembly/disassembly cycle of the COPI coat lattice on Golgi membranes. We found that both GBF1 and ArfGAP1 undergo fast exchange between Golgi membranes and cytosol. In the case of ArfGAP1, the extent of cytosol/Golgi exchange could be modulated by secretory cargo load, was independent of vesicle budding, and could be blocked in a coatomer-dependent fashion when Arf1 was permanently activated. These findings suggest that coatomer spatially localizes ArfGAP1 on membranes so that ArfGAP1’s catalytic activity is confined to Arf1 molecules in association with the COPI coat. The implication is that distinct Arf1-dependent coat/effector proteins recruit GAP proteins to different locations to hydrolyze GTP on Arf1. In the case of GBF1, its fast cytosol/membrane exchange means that its recruitment and that of Arf1 on membranes can be rapidly modulated. The drug brefeldin A (BFA), which acts as a noncompetitive inhibitor of the exchange reaction of GTP for GDP on Arf1, caused GBF1 to become stabilized on Golgi membranes, suggesting that a key target of BFA is GBF1 and that the drug leads to the formation of an Arf1-GBF1-BFA complex on membranes.
In mitosis, successful cell division depends on the coordination of chromosome, cytoskeleton, and organelle dynamics. We are currently investigating how such coordination occurs and have discovered a major role of Arf1, the small GTPase associated with the Golgi apparatus. Our data suggest that Arf1 helps orchestrate mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We found that, early in mitosis, Arf1 becomes inactive and dissociates from Golgi membranes, after which numerous Arf1-dependent peripheral Golgi proteins disperse. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of the enzyme’s GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. Such cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinesis. The findings suggest that mitotic Golgi disassembly is dependent on Arf1 inactivation and is used by the cell to disperse several peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis. We propose that Arf1 serves as a cell cycle regulator to coordinate Golgi dynamics with other cellular functions.
Altan-Bonnet N, Sougrat R, Lippincott-Schwartz J. Molecular basis for Golgi maintenance and biogenesis. Curr Opin Cell Biol 2004;16:364-372.
Altan-Bonnet N, Sougrat R, Liu W, Snapp EL, Ward T, Lippincott-Schwartz J. Golgi inheritance in mammalian cells is mediated through ER export activities. Mol Biol Cell 2005 [Epub ahead of print].
Liu W, Lippincott-Schwartz J. Illuminating COPII coat dynamics. Nat Struct Mol Biol2005;12:106-107.
Liu W, Moriyama K, Phair R, Duden R, Lippincott-Schwartz J. In vivo dynamics of ARFGAP1 and its functional interaction with Arf1 and coatomer on Golgi membranes. J Cell Biol 2005;168:1053-1063.
Niu T-K, Pfeifer AC, Lippincott-Schwartz J, Jackson CL. Dynamics of GBF1, a brefeldin A-sensitive Arf1 exchange factor at the Golgi. Mol Cell Biol 2005;16:1213-1222.
1Theresa Ward, PhD, former Visiting Fellow
2Erik Snapp, PhD, former Visiting Fellow
3Roman Polishchuk, PhD, former Visiting Fellow
4Anne Kenworthy, PhD, former Visiting Fellow
5Dave Frescas, BA, former Predoctoral Fellow
COLLABORATORS
Irwin Arias, MD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Federica Brandizzi, PhD, University of Saskatchewan, Saskatoon, Canada
Robert De Lotto, PhD, Kobenhavns Universitet, Copenhagen, Denmark
Ramanuhan Hegde, MD, PhD, Cell Biology and Metabolism Branch,NICHD, Bethesda, MD
Koret Hirschberg, PhD, Sackler School of Medicine, Tel Aviv University, Israel
Thomas Hodge, PhD, University of Georgia, Atlanta, GA
Cathy Jackson, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Mary Lilly, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Robert Phair, PhD, BioInformatics, Rockville, MD
Ronald Prywes, PhD, Columbia University, New York, NY
For further information, contact jlippin@helix.nih.gov.