Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB- sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.     

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.     

Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells

The internalization of a fluorescent analogue of phosphatidylethanolamine following its insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes composed of 50 mol % 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylethanolamine (C6-NBD-PE) and dioleoylphosphatidylcholine were incubated with monolayer cell cultures at 2 degrees C, a spontaneous transfer of the fluorescent lipid from liposomes to cells occurred. As long as the cells were kept at 2 degrees C, the fluorescent lipid remained at the plasma membrane. However, if, after removing the fluorescent liposomes, the cultures were warmed to 37 degrees C, the C6-NBD-PE was internalized and resided in the nuclear envelope, mitochondria, and Golgi apparatus in addition to the plasma membrane. Delivery of the fluorescent lipid to the Golgi apparatus could be blocked by the addition of 2-deoxyglucose plus sodium azide to the incubation medium. Evidence is presented suggesting that while delivery of the fluorescent lipid to the Golgi apparatus was mainly dependent on endocytosis, delivery to the nuclear envelope and mitochondria occurred by rapid transbilayer movement of the lipid across the plasma membrane followed by translocation of lipid monomers. Rapid transbilayer movement of C6-NBD-PE across the plasma membrane was found to be a temperature-dependent process that was blocked below 7 degrees C.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane.

Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin to acquire sphingolipids from the host within a few hours following infection. The transport of NBD-sphingolipid to the inclusion is energy- and temperature-dependent with the characteristics of an active, vesicle-mediated process. Photo-oxidation of C5-DMB-ceramide, in the presence of diaminobenzidine, identified DMB-lipids in vesicles in the process of fusing to the chlamydial inclusion membrane. C6-NBD-sphingomyelin incorporated into the plasma membrane is not trafficked to the inclusion to a significant degree, suggesting the pathway for sphingomyelin trafficking is direct from the Golgi apparatus to the chlamydial inclusion. Lectins and antibody probes for Golgi-specific glycoproteins demonstrate the close association of the chlamydial inclusion with the Golgi apparatus but do not detect these markers in the inclusion membrane. Collectively, the data are consistent with a model in which C.trachomatis inhabits a unique vesicle which interrupts an exocytic pathway to intercept host sphingolipids in transit from the Golgi apparatus to the plasma membrane.     

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.     

A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.     

The rate of bulk flow from the Golgi to the plasma membrane

A truncated analog of the backbone of sphingomyelin and glycolipids was synthesized. This truncated C8C8 ceramide was soluble in water (but was still able to cross cell membranes) and was utilized by the Golgi apparatus of living cells to produce water-soluble truncated phospholipids and glycolipids that were then secreted into the medium. Sphingomyelin is synthesized in a proximal (likely the cis) Golgi compartment. At 37 degrees C in CHO cells, the sphingomyelin analog is secreted with a half time of about 10 min. With this rate of bulk flow, no special signal is needed to pass through the Golgi to the plasma membrane. At 30 degrees C the half time of secretion of a lumenal ER marker is about 18 min, and that of the truncated sphingomyelin is about 14 min. Comparison of these rates sets an upper limit of about 4 min for half of the ER to be drained into the proximal Golgi at 30 degrees C.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes

This work describes two unusual features of membrane development in a eukaryotic cell. (a) The induction of an extensive network of tubovesicular membranes by the malaria parasite Plasmodium falciparum in the cytoplasm of the mature erythrocyte, and its visualization with two ceramide analogues C5-DMB-ceramide and C6-NBD-ceramide. "Sectioning" of the infected erythrocytes using laser confocal microscopy has allowed the reconstruction of detailed three-dimensional images of this novel membrane network. (b) The stage-specific export of sphingomyelin synthase, a biosynthetic activity concentrated in the Golgi of mammalian cells, to this tubovesicular network. Evidence is presented that in the extracellular merozoite stage the parasite retains sphingomyelin synthase within its plasma membrane. However, intracellular ring- and trophozoite-stage parasites export a substantial fraction (approximately 26%) of sphingomyelin synthase activity to membranes beyond their plasma membrane. Importantly we do not observe synthesis of new enzyme during these intracellular stages. Taken together these results strongly suggest that the export of this classic Golgi enzyme is developmentally regulated in Plasmodium. We discuss the significance of this export and the tubovesicular network with respect to membrane development and function in the erythrocyte cytosol.     

Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Synthesis and transport of different sphingomyelin species in rat Sertoli cells

Cellular phospholipids of Sertoli cells from immature rats were labeled with [¹⁴C]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [¹⁴C]-choline over a 45 h period of incubation demonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibility to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and SM2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease of about 60–70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-course experiments indicated a phosphatidylcholine SM precursor product relationship and differences in kinetic properties between SM1 and SM2. Resynthesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated that the resynthesized fraction reached the outer leaflet of the plasma membrane. The 60–70% inhibition of SM synthesis by monensin showed that the trans-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/medial Golgi apparatus. Additionally the results indicated that plasma membrane SM synthase activity could be the site of about 15% of SM synthesis in Sertoli cells.     

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.