Koret Hirschberg

ER-to-Golgi transport visualized in living cells

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.

Maintenance of Golgi structure and function depends on the integrity of ER export

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.

Golgi Membranes Are Absorbed into and Reemerge from the ER during Mitosis

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.

Coordinated Lipid Transfer between the Endoplasmic Reticulum and the Golgi Complex Requires the VAP Proteins and Is Essential for Golgi-mediated Transport

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.

Transport through the Golgi Apparatus by Rapid Partitioning within a Two-Phase Membrane System

The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already in the system and exit from partitioned domains with no cargo privileged for export based on its time of entry into the system. Given these results, we constructed a new model of intra-Golgi transport that involves rapid partitioning of enzymes and transmembrane cargo between two lipid phases combined with relatively rapid exchange among cisternae. Simulation and experimental testing of this rapid partitioning model reproduced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves.

Microtubules Support Production of Starvation-induced Autophagosomes but Not Their Targeting and Fusion with Lysosomes

Autophagy is a major catabolic pathway in eukaryotic cells whereby the lack of amino acids induces the formation of autophagosomes, double-bilayer membrane vesicles that mediate delivery of cytosolic proteins and organelles for lysosomal degradation. The biogenesis and turnover of autophagosomes in mammalian cells as well as the molecular mechanisms underlying induction of autophagy and trafficking of these vesicles are poorly understood. Here we utilized different autophagic markers to determine the involvement of microtubules in the autophagic process. We show that autophagosomes associate with microtubules and concentrate near the microtubule-organizing center. Moreover, we demonstrate that autophagosomes, but not phagophores, move along these tracks en route for degradation. Disruption of microtubules leads to a significant reduction in the number of mature autophagosomes but does not affect their life span or their fusion with lysosomes. We propose that microtubules serve to deliver only mature autophagosomes for degradation, thus providing a spatial barrier between phagophores and lysosomes.

Involvement of Secreted Aspergillus fumigatus Proteases in Disruption of the Actin Fiber Cytoskeleton and Loss of Focal Adhesion Sites in Infected A549 Lung Pneumocytes

Aspergillus fumigatus is an opportunistic pathogenic fungus that predominantly infects the respiratory system. Penetration of the lung alveolar epithelium is a key step in the infectious process. The cytoskeleton of alveolar epithelial cells forms the cellular basis for the formation of a physical barrier between the cells and their surroundings. This study focused on the distinct effects of A. fumigatus on the actin cytoskeleton of A549 lung pneumocytes. Of the 3 major classes of cytoskeletal fibers--actin microfilaments, microtubules, and intermediate filaments--only the actin cytoskeleton was found to undergo major structural changes in response to infection, including loss of actin stress fibers, formation of actin aggregates, disruption of focal adhesion sites, and cell blebbing. These changes could be specifically blocked in wild-type strains of A. fumigatus by the addition of antipain, a serine and cysteine protease inhibitor, and were not induced by an alkaline serine protease-deficient strain of A. fumigatus. Antipain also reduced, by approximately 50%, fungal-induced A549 cell detachment from the plates and reduction in viability. Our findings suggest that A. fumigatus breaches the alveolar epithelial cell barrier by secreting proteases that act together to disorganize the actin cytoskeleton and destroy cell attachment to the substrate by disrupting focal adhesions.

MONITORING THE DYNAMICS AND MOBILITY OF MEMBRANE PROTEINS TAGGED WITH GREEN FLUORESCENT PROTEIN

Publisher Summary This chapter discusses the methods that can be used to study intracellular membrane dynamics using green fluorescent protein (GFP)-tagged membrane proteins expressed in living cells. The use of GFP chimeras overcomes many of the limitations inherent to the studies of fixed specimens labeled with antibodies. Not only can GFP chimeras be visualized in the unperturbed environment of a living cell, virtually any protein can be tagged with GFP, producing fusion proteins that are intrinsically fluorescent and photostable and that usually retain parent molecule targeting and function. The chapter discusses approaches for optimizing GFP chimera brightness and expression levels within cells, so that intracellular membranes expressing GFP constructs can be readily resolved. The chapter also discusses the techniques for acquiring time-lapse images and methods to analyze them. Approaches for quantitating the number of GFP molecules being imaged in a cell have been described, so that the kinetic steps of membrane transport can be analyzed. The chapter describes the methods of photobleaching that can be used to measure the diffusional mobility of GFP chimeras and the extent of their movement between membrane-bound compartments.

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

MAL, a compact hydrophobic, four-transmembrane-domain apical protein that copurifies with detergent-resistant membranes is obligatory for the machinery that sorts glycophosphatidylinositol (GPI)-anchored proteins and others to the apical membrane in epithelia. The mechanism of MAL function in lipid-raft-mediated apical sorting is unknown. We report that MAL clusters formed by two independent procedures-spontaneous clustering of MAL tagged with the tandem dimer DiHcRED (DiHcRED-MAL) in the plasma membrane of COS7 cells and antibody-mediated cross-linking of FLAG-tagged MAL-laterally concentrate markers of sphingolipid rafts and exclude a fluorescent analogue of phosphatidylethanolamine. Site-directed mutagenesis and bimolecular fluorescence complementation analysis demonstrate that MAL forms oligomers via xx intramembrane protein-protein binding motifs. Furthermore, results from membrane modulation by using exogenously added cholesterol or ceramides support the hypothesis that MAL-mediated association with raft lipids is driven at least in part by positive hydrophobic mismatch between the lengths of the transmembrane helices of MAL and membrane lipids. These data place MAL as a key component in the organization of membrane domains that could potentially serve as membrane sorting platforms.

Role for TBC1D20 and Rab1 in Hepatitis C Virus Replication via Interaction with Lipid Droplet-Bound Nonstructural Protein 5A

ABSTRACT Replication and assembly of hepatitis C virus (HCV) depend on the hosts secretory and lipid-biosynthetic machinery. Viral replication occurs on endoplasmic reticulum (ER)-derived modified membranes, while viral assembly is thought to occur on lipid droplets (LDs). A physical association and coordination between the viral replication and assembly complexes are prerequisites for efficient viral production. Nonstructural protein 5A (NS5A), which localizes both to the ER and LDs, is an ideal candidate for this function. Here, the interaction of NS5A with host cell membranes and binding partners was characterized in living cells. The binding of NS5A to LDs is apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV-infected cells, NS5A fluorescence was observed around the LDs and in perinuclear structures that were incorporated into a highly immobile platform superimposed over the ER membrane. Moreover, TBC1D20 and its cognate GTPase Rab1 are recruited by NS5A to LDs. The NS5A-TBC1D20 interaction was shown to be essential for the viral life cycle. In cells, expression of the Rab1 dominant negative (Rab1DN) GTPase mutant abolished steady-state LDs. In infected cells, Rab1DN induced the elimination of NS5A from viral replication sites. Our results demonstrate the significance of the localization of NS5A to LDs and support a model whereby its interaction with TBC1D20 and Rab1 affects lipid droplet metabolism to promote the viral life cycle.