The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.     

Cultured oligodendrocytes mimic in vivo phenotypic characteristics: Cell shape, expression of myelin-specific antigens, and membrane production

Primary cultures of neonatal mouse cerebra were maintained for up to 4 weeks in the absence of neurons. Oligodendrocytes in these cultures pass through a sequence of cytoarchitectural change and antigen expression which mimics the differentiation of oligodendrocytes in vivo. The cell bodies and processes of oligodendrocytes first express the myelin-specific antigen galactocerebroside (GC) by 2 days in vitro. Myelin basic protein (MBP) appears several days later. The majority of oligodendrocytes then proceed to elaborate large sheets of membranous material from the tips and lengths of cell processes. These membranous sheets, which contain GC and MBP, are reminiscent of unwrapped myelin profiles in vivo. As with the cell bodies and processes, GC is inserted into the sheets several days before MBP. Our results establish that oligodendrocytes cultured without neurons are able to produce extensive membranes containing myelin-specific antigens. They also suggest that oligodendrocyte shape and membrane production are, in part, regulated from within the oligodendrocyte itself.     

Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

Myelin basic protein (MBP) mRNA is localized to myelin produced by oligodendrocytes of the central nervous system. MBP mRNA microinjected into oligodendrocytes in primary culture is assembled into granules in the perikaryon, transported along the processes, and localized to the myelin compartment. In this work, microinjection of various deleted and chimeric RNAs was used to delineate regions in MBP mRNA that are required for transport and localization in oligodendrocytes. The results indicate that transport requires a 21-nucleotide sequence, termed the RNA transport signal (RTS), in the 3′ UTR of MBP mRNA. Homologous sequences are present in several other localized mRNAs, suggesting that the RTS represents a general transport signal in a variety of different cell types. Insertion of the RTS from MBP mRNA into nontransported mRNAs, causes the RNA to be transported to the oligodendrocyte processes. Localization of mRNA to the myelin compartment requires an additional element, termed the RNA localization region (RLR), contained between nucleotide 1,130 and 1,473 in the 3′ UTR of MBP mRNA. Computer analysis predicts that this region contains a stable secondary structure. If the coding region of the mRNA is deleted, the RLR is no longer required for localization, and the region between nucleotide 667 and 953, containing the RTS, is sufficient for both RNA transport and localization. Thus, localization of coding RNA is RLR dependent, and localization of noncoding RNA is RLR independent, suggesting that they are localized by different pathways.     

Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.     

A size barrier limits protein diffusion at the cell surface to generate lipid-rich myelin-membrane sheets

The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.     

SNARE protein trafficking in polarized MDCK cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.     

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Cell polarity in myelinating glia: from membrane flow to diffusion barriers

Myelin-forming glia are highly polarized cells that synthesize as an extension of their plasma membrane, a multilayered myelin membrane sheath, with a unique protein and lipid composition. In most cells polarity is established by the polarized exocytosis of membrane vesicles to the distinct plasma membrane domains. Since myelin is composed of a stack of tightly packed membrane layers that do not leave sufficient space for the vesicular trafficking, we hypothesize that myelin does not use polarized exocytosis as a primary mechanism, but rather depends on lateral transport of membrane components in the plasma membrane. We suggest a model in which vesicle-mediated transport is confined to the cytoplasmic channels, from where transport to the compacted areas occurs by lateral flow of cargo within the plasma membrane. A diffusion barrier that is formed by MBP and the two adjacent cytoplasmic leaflets of the myelin bilayers acts a molecular sieve and regulates the flow of the components. Finally, we highlight potential mechanism that may contribute to the assembly of specific lipids within myelin. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.     

Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.     

Expression of Myelin Protein Genes and Other Myelin Components in an Oligodendrocytic Cell Line Conditionally Immortalized with a Temperature-Sensitive Retrovirus

Abstract: We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2′,3′-cyclic nucleotide 3′-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34°C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39°C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39°C for >9 days compared with cells maintained at 34°C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers—galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a “mature” oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of “maturation’. This cell line has now been passaged >40 times with fidelity of phenotype and genotype.     

Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells.

Syntaxins are cytoplasmically oriented integral membrane soluble NEM-sensitive factor receptors (SNAREs; soluble NEM-sensitive factor attachment protein receptors) thought to serve as targets for the assembly of protein complexes important in regulating membrane fusion. The SNARE hypothesis predicts that the fidelity of vesicle traffic is controlled in part by the correct recognition of vesicle SNAREs with their cognate target SNARE partner. Here, we show that in the exocrine acinar cell of the pancreas, multiple syntaxin isoforms are expressed and that they appear to reside in distinct membrane compartments. Syntaxin 2 is restricted to the apical plasma membrane whereas syntaxin 4 is found most abundantly on the basolateral membranes. Surprisingly, syntaxin 3 was found to be localized to a vesicular compartment, the zymogen granule membrane. In addition, we show that these proteins are capable of specific interaction with vesicle SNARE proteins. Their nonoverlapping locations support the general principle of the SNARE hypothesis and provide new insights into the mechanisms of polarized secretion in epithelial cells.     

Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24-29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.     

SncRNA715 Inhibits Schwann Cell Myelin Basic Protein Synthesis

Myelin basic proteins (MBP) are major constituents of the myelin sheath in the central nervous system (CNS) and the peripheral nervous system (PNS). In the CNS Mbp translation occurs locally at the axon-glial contact site in a neuronal activity-dependent manner. Recently we identified the small non-coding RNA 715 (sncRNA715) as a key inhibitor of Mbp translation during transport in oligodendrocytes. Mbp mRNA localization in Schwann cells has been observed, but has not been investigated in much detail. Here we could confirm translational repression of Mbp mRNA in Schwann cells. We show that sncRNA715 is expressed and its levels correlate inversely with MBP in cultured Schwann cells and in the sciatic nerve in vivo. Furthermore we could reduce MBP protein levels in cultured Schwann cells by increasing the levels of the inhibitory sncRNA715. Our findings suggest similarities in sncRNA715-mediated translational repression of Mbp mRNA in oligodendrocytes and Schwann cells.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

Comparative localization of Wolfgram W1 and myelin basic proteins in the rat brain during ontogenesis

Summary Antisera raised in rabbits against myelin basic proteins (MBP) and Wolfgram W1 protein isolated from rat myelin were used to study the maturation of oligodendrocytes in the developing rat nervous system. Both proteins were localized immunohistochemically at the light and electron microscopical levels in rat brain from the time of their first appearance to the adult stage. Oligodendrocytes were first detected by their positive staining with W1 antiserum two days after birth and at 1–3 days later with MBP antiserum. At 8–10 days, the number of oligodendrocytes labelled with both sera increases and the myelinated fibre pathways were clearly visible. Labelling with W1 antiserum was observed in oligodendrocytes at all stages from 2 days after birth to adulthood and in myelin fibres when they were present. In contrast, staining of oligodendroglial cells with MBP declined during the period of rapid myelination (20–25 days after birth) and finally disappeared, whereas myelin staining was still apparent. The electron microscopical study revealed that the synthesis of Wolfgram proteins occurred mostly at the peripheral cytoplasmic ribosomes of the cells, from where they were probably transported to processes engaged in myelination. The electron micrographs also showed that the sites of MBP synthesis seemed to be more uniformly distributed over the entire cytoplasm.