Campylobacter jejuni--microtubule-dependent invasion.

Campylobacter jejuni is the leading bacterial cause of food-borne illness worldwide and a major cause of Guillain-Barré paralysis. Recent molecular and cellular studies of one well-characterized C. jejuni strain have begun to unravel the details of an unusual microtubule-dependent (actin-filament-independent) gut-invasion mechanism, through which at least some C. jejuni initiate disease. Although responsible for causing a human dysenteric syndrome remarkably similar to that triggered by Shigella spp., current evidence suggests that C. jejuni use some markedly different molecular mechanisms of pathogenesis compared with shigellae.

Golgi complex reorganization during muscle differentiation: visualization in living cells and mechanism.

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1-2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.

Cell cycle maintenance and biogenesis of the Golgi complex.

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.

Dynamics and retention of misfolded proteins in native ER membranes.

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.

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.

Actin filaments and microtubules are involved in different membrane traffic pathways that transport sphingolipids to the apical surface of polarized HepG2 cells.

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked "hyperpolarization," i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?

A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2. A 125I-labeled, photoreactive ceramide, 125I-N3-Cer, was incubated with the cells and became incorporated into two sphingolipid products. The major product was photoreactive sphingomyelin (125I-N3-SM) (25% of total radioactivity), while only minor amounts of photoreactive glucosylceramide (125I-N3-GlcCer) were formed (< 2%). After photoactivation, a restricted number of proteins was labeled. Given the absolute amounts of the newly synthesized, photoreactive lipids and their precursor present in the cells, labeling of the proteins can be assumed to be derived from interaction with either ceramide (Cer) or sphingomyelin (SM), or both. To discriminate between these possibilities, photoactivation and protein analysis was performed in cells treated with D-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol (PDMP), an inhibitor of sphingolipid biosynthesis. In treated cells, the radioactive SM pool was reduced by approximately 80%. Concomitantly, labeling of a 60-kd protein, seen in control cells, decreased. Furthermore, the 60-kd protein is membrane-associated and insoluble in detergent at low temperature. Moreover, when cells containing photoreactive sphingolipids after a preincubation with the photoreactive Cer were photoactivated and subsequently incubated with fluorescent sphingolipid analogs, transport of the latter to the bile canalicular membrane, as observed in control cells, was inhibited. Taken together, the data suggest that distinct proteins, among them a 60-kd protein, may play a specific and functional role in sphingolipid transport to the bile canalicular membrane.

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.

Golgi tubule traffic and the effects of brefeldin A visualized in living cells.

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein-Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5-10 min until rapidly emptying Golgi contents into the ER within 15-30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

Intracellular sites involved in the biogenesis of bile canaliculi in hepatic cells.

Studies in hepatoma cells and hepatocytes have revealed that the biogenesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well differentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to cell surface contact sites of attached cells. We demonstrate that a fluorescent head group-labeled lipid analog, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertion into the plasma membrane of HepG2 cells at 4 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that the MLV are a target for plasma membrane-derived lipids. Furthermore, also the Golgi apparatus is involved in the formation of the vesicles. After initial accumulation of the fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (C6-NBD)-ceramide in the Golgi apparatus at 37 degrees C, prolonged incubation at 37 degrees C results in the appearance of NBD fluorescence in the microvilli-lined vesicles. The transport route for the Golgi-derived material to the developing bile canalicular vesicle is not an indirect pathway, i.e. involving transcytosis via the basolateral plasma membrane. This could be demonstrated by including bovine serum albumin (BSA) in the incubation media, a lipid scavenger that will remove any C6-NBD-lipids exposed at the basolateral membrane. At these conditions, lipid trafficking between the Golgi complex and MLV still occurred. We further demonstrate that the targeting from the Golgi apparatus to the bile canaliculus is also operational in isolated human hepatocytes. The latter results suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully differentiated cells.

Processing of the phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine by rat hepatocytes in vitro and in vivo.

We have investigated the processing of the non-exchangeable fluorescent phospholipid analogue phosphatidyl(N-sulphorhodamine B sulphonyl)ethanolamine (N-Rh-PE) by rat liver cells. In the hepatocyte couplet system, N-Rh-PE was incorporated into the plasma membrane at 2 degrees C and readily internalized upon warming to 37 degrees C. Fluorescence was initially found to be concentrated in vesicles clustered throughout the cell, but subsequently it started to accumulate in pericanalicular vesicles, tentatively identified as lysosomes, and in the bile canalicular lumen. Analysis of cells and media by t.l.c. revealed the slow formation of at least two metabolites. After intravenous injection into bile-fistula rats of [9,10-3H-oleoyl]N-Rh-PE incorporated in small unilamellar liposomes, the initial rates of elimination from plasma of 3H and rhodamine label were virtually identical. However, biliary secretion of the 3H label (5.5% of dose at 2 h) was much slower than that of the rhodamine label (49.3% at 2 h). The rhodamine label in bile was chloroform-soluble, but not identical to the native molecule, and was resistant to phospholipase A2 and alkaline hydrolysis. To gain insight in the mechanism of the rapid bile secretion of this metabolite, we compared the processing of N-Rh-PE, its deacylated form [glycerophospho(N-sulphorhodamine B sulphonyl)ethanolamine; Gly-N-Rh] and the rhodamine label itself (sulphorhodamine B sulphonyl chloride; SRho). Intravenous injection of chloroform-soluble N-Rh-PE and of methanol/water-soluble Gly-N-Rh complexed with albumin both resulted in rapid bile secretion of chloroform-soluble fluorescent compounds (60.2% and 86.3% respectively at 2 h), which showed behaviour identical to that of the metabolite of liposomal N-Rh-PE on t.l.c. Methanol/water-soluble SRho was also rapidly secreted into bile (89.5% at 2 h) without being metabolized. Bile secretion of the chloroform-soluble metabolite of N-Rh-PE and of SRho was markedly impaired (-31% and -52% respectively) in GY Wistar rats, which express a genetic defect in the hepatobiliary transport of organic anions. Our data show that the rat hepatocyte is capable of modifying the structure of N-Rh-PE, a process which proceeds considerably faster in vivo than in vitro. The chloroform-soluble metabolite is subsequently rapidly removed via the bile. The canalicular organic anion transporting system, which is deficient in GY rats, appears to be involved in the excretion of this apolar product of hepatic metabolism.

Autographa californica M nuclear polyhedrosis virus: microtubules and replication.

Progressive reorganization and depolymerization of microtubules corresponded with virus-induced rounding of Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected Spodoptera frugiperda IPLB-Sf-21 cells, suggesting that microtubules were instrumental in maintaining the normal shape of these cells. Depolymerization of all cortical and most of the paranuclear microtubules with colchicine also resulted in cell rounding, confirming this hypothesis. Studies with aphidicolin and cycloheximide indicated the virus-induced effects on the microtubules were mediated by both early and late viral gene products. Microtubules in cells infected with a p10 deletion mutant depolymerized microtubules in a manner similar to those in wild-type virus-infected cells, indicating p10 was not responsible for virus-induced changes in the microtubules. Nevertheless, evidence for the association of p10 and microtubules was obtained by fluorescence microscopy and immunoelectron microscopy. Colchicine depolymerization of microtubules before and throughout infection did not interfere with virus replication, but treatment of cells with taxol, a microtubule-stabilizing agent, both delayed and depressed virus replication. The taxol-induced effect was relieved by the addition of colchicine. These results suggested that AcMNPV-induced depolymerization of microtubules may be a necessary event in, rather than a tangential effect of, virus replication. Attempts to monitor the effects of virus infection on intermediate filaments were unsuccessful due to the lack of cross-reactivity between antibodies to intermediate filament proteins and IPLB-Sf-21 cells, indicating these proteins are not highly conserved in lepidopteran insect cells.

Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene.

The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene. The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA. Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10. Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes. Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent. The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells. Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product. The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane. The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S. exigua larvae than was wild-type AcMNPV. The increased virulence of the recombinant is an important property for the control of insects.