Dissecting the molecular mechanisms of polarized membrane traffic: reconstitution of three transport steps in epithelial cells using streptolysin-O permeabilization.
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Animals↗
Biomedical subjects
K Simons
Publications and source records attributed to K Simons.
rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.
Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.
Actins↗
Nocodazole-dependent transport, and brefeldin A--sensitive processing and sorting, of newly synthesized membrane proteins in cultured neurons.
The envelope glycoproteins of Semliki Forest virus (SFV), Vesicular Stomatitis virus (VSV), and Influenza Fowl Plague virus (FPV) are vectorially targeted in neurons to the plasma membrane of dendrites (SFV and VSV) and axons (FPV). To gain insight into the mechanisms responsible for such polarized delivery we have examined the effects on neurons of nocodazole and brefeldin A (BFA), which are known to cause microtubule depolymerization and disassembly of the Golgi apparatus, respectively. Nocodazole treatment blocked transport of all viral glycoproteins to both axons and dendrites. BFA treatment induced disruption of the Golgi complex, including the trans-Golgi network (TGN), and tubulation of endosomes. However, the delivery of the SFV and FPV glycoproteins to the cell surface was not affected significantly by BFA, although processing and sorting were altered, as revealed by surface biotinylation and immunofluorescence microscopy of fixed nonpermeabilized cells. These results demonstrate the involvement of microtubules in axonal and dendritic transport of integral membrane glycoproteins, and the existence of a BFA-sensitive component in the sorting but not in the transport machinery.
Animals↗
Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae.
Antibody binding to glycolipids and glycophosphatidylinositol (GPI)-anchored proteins of lymphocytes can trigger activation of specific signal transduction pathways. The finding that GPI-anchored proteins are present in detergent-insoluble complexes with several tyrosine kinases of the Src family suggested that these complexes may represent membrane microdomains involved in the transduction of signals to the cell interior. Recent work has suggested a link between detergent-insoluble microdomains and plasma membrane invaginations termed caveolae. Here we show that lymphocytes lack plasma membrane domains with the characteristic features of caveolae. Furthermore, VIP21-caveolin was not detectable in four different lymphocyte cell lines at the protein or mRNA level. In addition to the lack of caveolar domains, capping experiments suggested that the bulk of the GPI-anchored protein Thy1 and the glycosphingolipid GM1 were not stably associated in the lymphocyte plasma membrane. Despite this, Thy1 and GM1 were present in detergent-insoluble complexes. We conclude that detergent insolubility does not correlate with the presence of caveolae or of VIP21-caveolin and that caveolae, as defined by a number of different markers, are not involved in signal transduction in lymphocytes.
3T3 Cells↗
Activators of protein kinase A stimulate apical but not basolateral transport in epithelial Madin-Darby canine kidney cells.
In polarized Madin-Darby canine kidney cells the newly synthesized plasma membrane proteins, on the exocytic pathway, are sorted in the trans-Golgi network (TGN) and delivered directly to the apical or basolateral surface. Forskolin, isobutylmethylxanthine, and dibutyryl cAMP, all known to activate protein kinase A, stimulated transport of influenza hemagglutinin (HA) from the TGN to the apical surface. The same reagents, however, did not affect the transport of HA from the endoplasmic reticulum to the Goli complex nor did they affect transport of vesicular stomatitis virus G protein from the TGN to the basolateral surface. The addition of staurosporin, a general protein kinase inhibitor, did not affect the transport of HA in nontreated cells but blocked the stimulation caused by the above reagents. Apical transport of HA was also stimulated by phorbol ester, an activator of protein kinase C. Activation of apical transport by phorbol ester as well as aluminum fluoride (Pimplikar, S. W., and Simons, K. (1993) Nature 362, 456-458) was also negated by staurosporin. These results show that in polarized Madin-Darby canine kidney cells, protein kinase A and protein kinase C selectively stimulate the apical transport.
Alkaloids↗
Myelin membrane biogenesis by oligodendrocytes. Developmental regulation of low molecular weight GTP-binding proteins.
Oligodendrocytes synthesize dramatic amounts of myelin membrane. We hypothesized that this requires unique aspects of vesicular trafficking. Specific stages of the oligodendrocyte lineage were assayed for low molecular weight GTP-binding proteins implicated in the regulation of vesicular transport pathway (two dimensional gel electrophoresis, [alpha-32P]GTP overlay). Consistent with the hypothesis, as oligodendrocytes differentiate from early progenitors to mature myelin-producing cells, > or = 12 small GTP-binding proteins become up-regulated. Myelin membrane also has a complex pattern of GTP-binding proteins. Several of these proteins may be specific to oligodendrocytes, suggesting that oligodendrocytes may utilize cell-type specific GTP-binding proteins for biogenesis and maintenance of the myelin membrane.
Animals↗
A putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins.
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Amino Acid Sequence↗
VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.
In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.
Amino Acid Sequence↗
Isolation of a mouse cDNA encoding Rab23, a small novel GTPase expressed predominantly in the brain.
The full-length cDNA encoding Rab23, a novel Ras-related small GTPase, was isolated using the sequence of a previously described [Chavrier et al., Gene 112 (1992) 261-264] short cDNA fragment and the rapid amplification of cDNA ends (RACE) PCR techniques. The deduced amino acid sequence was not very closely related to any previously described small GTPase, but was within the Rab subfamily. A Northern analysis revealed that the rab23 mRNA is predominantly expressed in the brain, which places the protein, together with Rab3a and Rab15, in the group of small GTPases characteristic of the nervous system.
Amino Acid Sequence↗
Expression of exogenous proteins in mammalian cells with the Semliki Forest virus vector.
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Animals↗
Color dissociation artifacts in double Maddox rod cyclodeviation testing.
BACKGROUND: The double Maddox rod test, based on a red Maddox rod in front of one eye and a clear Maddox rod in front of the other, is used to measure cyclodeviation, typically in patients with superior oblique muscle pareses. Discrepant results between the double Maddox rod test and other torsion measures, and reports of "paradoxic" cyclodeviation in the normal eye of some patients with superior oblique paresis, suggest the two-color format of the double Maddox rod test may produce artifactual torsion measures. METHODS: Forty patients with superior oblique paresis were tested twice using the double Maddox rod test, reversing the red and white Maddox rods between eyes for the second test, and 18 were tested further with same-color red or clear Maddox rods in front of both eyes. RESULTS: With the standard double Maddox rod test, 33 (83%) of 40 patients localized their cyclodeviation to the eye viewing through the red Maddox rod, irrespective of laterality of the paresis or fixation preference. In all 33 patients, laterality of the perceived torsion changed between eyes when testing was repeated with red and white Maddox rods interchanged between eyes. With same-color Maddox rods before both eyes, 17 (94%) of 18 patients localized extorsion to the paretic eye. There was 7.6:1 ratio of luminance transmission and a 1.6:1 ratio of grating spatial frequency bandpass in the plano meridian between the clear and red Maddox rods, which appear to be responsible for the double Maddox rod test artifact. CONCLUSION: The traditional double Maddox rod test may produce artifactual cyclodeviation measurements. An alternative version of the test, based on same-color Maddox rods in front of both eyes, is proposed. The relatively high spatial frequency bandpass characteristics of the plano meridian of the Maddox rod (as high as 20/25 Snellen equivalent resolution through the clear Maddox rod) also suggests double Maddox rod testing should be conducted in a dark room to avoid biases from visual environment cues.
Artifacts↗
Basolateral protein transport in streptolysin O-permeabilized MDCK cells.
We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.
Amino Acid Sequence↗
Regulated internalization of caveolae.
Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.
Alkaline Phosphatase↗
Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.
Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.
Amino Acid Sequence↗
Expression of heterologous proteins in cultured rat hippocampal neurons using the Semliki Forest virus vector.
The Semliki Forest virus expression vector (Liljeström and Garoff: Bio/Technology 9:1356-1361, 1991) was tested in cultured rat hippocampal neurons using two Madin-Darby canine kidney (MDCK) cell membrane-associated proteins as reporters: rab8, a small GTPase involved in post-Golgi vesicle transport, and VIP21, an integral membrane protein of caveolae, trans-Golgi network, and post-Golgi vesicles. Expression of the c-myc epitope-tagged proteins was visualized by immunofluorescence microscopy. The proteins were first detected in neurons after 3-4 hr infection by the recombinant viruses. The infection efficiency on neurons was high: after 6 hr infection at a multiplicity of one, 50-60% of the cells expressed the reporter proteins. The neurons tolerated the infection well up to 8 hr. Their polarized organization was not disturbed, as judged from morphology and from distribution of the dendritic MAP2 and axonal synaptophysin marker proteins. The Semliki Forest virus vector thus seems suitable for short-term expression of proteins in cultured neurons.
Amino Acid Sequence↗
Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells.
In simple epithelial cells, the delivery of apical and basolateral proteins to the cell surface is mediated by sorting in the trans-Golgi network and transport via separate vesicular carriers. In order to identify the molecular machinery involved in protein sorting, we have recently studied a detergent-insoluble complex in Madin-Darby canine kidney (MDCK) cells, following CHAPS extraction of exocytic carrier vesicles, specifically including the apical marker protein influenza hemagglutinin (HA). Previously, a Triton X-100 insoluble membrane residue that was enriched in glycosylphosphatidylinositol-anchored (GPI) proteins and glycolipids was characterized and implicated in transport to the apical cell surface [Brown, D., & Rose, J. (1991) Cell 68, 533-544]. In this report, the protein compositions of the CHAPS and Triton complexes have been compared by two-dimensional gel analysis. Only a few major membrane proteins are found in the complexes. The protein compositions are qualitatively similar, but differ quantitatively in the individual components. The CHAPS complex is depleted of GPI-linked proteins and retains a minor fraction of lipids similar in composition to that of the Triton X-100 insoluble complex. We propose that in vivo the complexes form part of a sorting platform that mediates protein segregation and delivery to the apical cell surface.
Animals↗
Polarized distribution of the viral glycoproteins of vesicular stomatitis, fowl plague and Semliki Forest viruses in hippocampal neurons in culture: a light and electron microscopy study.
We have shown previously using immunofluorescence microscopy that upon infection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface (Cell, 62 (1990) 63-72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusively present in the dendrites and cell body but not in axons.
Animals↗
Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein.
The role of heterotrimeric GTP-binding proteins in signal transduction is well established. They might also be involved in vesicular transport. Here we show that in the epithelial cell line Madin-Darby Canine Kidney, transport of influenza haemagglutinin protein to the apical surface is stimulated and that of vesicular stomatitis virus glycoprotein to the basolateral surface is retarded by AlF(3-5) treatment. Treatment of cells with the reagents known to influence the Gi class of G proteins affected only the basolateral pathway whereas reagents acting on the Gs class of G proteins specifically affected the apical pathway. In permeabilized cells, antibodies raised against the N-terminal domain of the alpha-subunit of Gs inhibited the transport of haemagglutinin from the trans-Golgi network to apical surface but not between the endoplasmic reticulum and Golgi complex. These observations demonstrate involvement of a Gs class of heterotrimeric G proteins, besides that of the Gi, in vesicular transport. Moreover, the apical and the basolateral pathways in epithelial cells seem to be regulated by Gs and Gi proteins, respectively, in the trans-Golgi network.
Animals↗