Search PubMed⌕ Search

Biomedical subjects

K Simons

Publications and source records attributed to K Simons.

At least 73 records · Page 4Linked to original sources
PubMed1996

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells.

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.

Aluminum Compounds↗
PubMed1996

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

Actin Cytoskeleton↗
PubMed1996

Roles for Rac1 and Cdc42 in planar polarization and hair outgrowth in the wing of Drosophila.

The wing of Drosophila melanogaster is covered by an array of distally pointing hairs. A hair begins as a single membrane outgrowth from each wing epithelial cell, and its distal orientation is determined by the restriction of outgrowth to a single distal site on the cell circumference (Wong, L., and P. Adler. 1993. J. Cell Biol. 123:209-211.). We have examined the roles of Cdc42 and Rac1 in the formation of wing hairs. We find that Cdc42 is required for localized actin polymerization in the extending hair. Interfering with Cdc42 activity by expression of a dominant negative protein abolishes both localized actin polymerization and hair outgrowth. In contrast, Rac1 is important for restricting the site at which hairs grow out. Cells expressing the dominant negative Rac1N17 fail to restrict outgrowth to a single site and give rise to multiple wing hairs. This polarity defect is associated with disturbances in the organization of junctional actin and also with disruption of an intricate microtubule network that is intimately associated with the junctional region. We also find that apical junctions and microtubules are involved in structural aspects of hair outgrowth. During hair formation, the apical microtubules that point distally elongate and fill the emerging wing hair. As the hair elongates, junctional proteins are reorganized on the proximal and distal edges of each cell.

Actin Cytoskeleton↗
PubMed1996

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.

Animals↗
PubMed1996

Characterization of VIP36, an animal lectin homologous to leguminous lectins.

VIP36 was isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes. The protein is localized to the Golgi apparatus and the cell surface, and belongs to a new family of legume lectin homologues in the animal secretory pathway that might be involved in the trafficking of glycoproteins, glycolipids or both. Here we show that VIP36 is N-glycosylated and expressed in organs abundant in epithelial cells as well as in non-epithelial organs. Our studies demonstrate that the recombinant exoplasmic/luminal domain of VIP36 binds Ca2+ and that the protein decorates internal membrane structures of MDCK cells in vitro that are distinct from the Golgi apparatus. This binding requires Ca2+ and can be specifically inhibited by N-acetyl-D-galactosamine. The recombinant protein was used for affinity chromatography. Glycopeptides obtained from [3H]galactose-labelled cells bind to VIP36 and can be eluted with N-acetyl-D-galactosamine. Our data imply that VIP36 functions as a lectin in post-Golgi trafficking.

3T3 Cells↗
PubMed1995

VIP17/MAL, a proteolipid in apical transport vesicles.

VIP17 is a proteolipid enriched in the CHAPS-insoluble complexes from MDCK cells, and a candidate component of the molecular machinery responsible for the sorting and targeting of proteins to the apical surface. Cloning and sequencing of the cDNA encoding the protein revealed that it is the canine homolog of the human and rat MAL proteins. Analysis by immunofluorescence microscopy of epitope-tagged VIP17/MAL expressed transiently in BHK cells and stably in MDCK cells revealed a perinuclear, vesicular, and plasmalemmal staining. In MDCK cells the distribution was mainly in vesicular structures in the apical cytoplasm. These and other results suggest that VIP17/MAL is an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane.

Amino Acid Sequence↗
PubMed1995

A photo-reactive derivative of ganglioside GM1 specifically cross-links VIP21-caveolin on the cell surface.

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo-reactive cross-linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross-linked by the GM1 derivative was VIP21-caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21-caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.

Animals↗
PubMed1995

N-glycans as apical sorting signals in epithelial cells.

In epithelial Madin-Darby canine kidney (MDCK) cells newly synthesized molecules are sorted in the trans-Golgi network and directly delivered to their apical and basolateral surface destinations. Sorting is mediated by signals in the cytoplasmic domains of basolateral transmembrane proteins whereas glycosylphosphatidylinositol-linked proteins have apical sorting information in their glycolipid tails. Signals for apical transmembrane proteins are thought to reside in their ectodomains, because truncated forms lacking the cytoplasmic tail and the membrane anchor are secreted apically. Here we demonstrate that carbohydrates act as an apical targeting signal for secretory proteins. Growth hormone, which is non-glycosylated and secreted from both sides of MDCK cell layers, is secreted from the apical side when glycosylated. Thus glycans not only play a general role in protein folding but also appear to function in protein sorting in biosynthetic traffic.

Animals↗
PubMed1995

VIP21/caveolin is a cholesterol-binding protein.

VIP21/caveolin is localized to both caveolae and apical transport vesicles and presumably cycles between the cell surface and the Golgi complex. We have studied the lipid interactions of this protein by reconstituting Escherichia coli-expressed VIP21/caveolin into liposomes. Surprisingly, the protein reconstituted only with cholesterol-containing lipid mixtures. We demonstrated that the protein binds at least 1 mol of cholesterol per mole of protein and that this binding promotes formation of protein oligomers. These findings suggest that VIP21/caveolin, through its cholesterol-binding properties, serves a specific function in microdomain formation during membrane trafficking.

Animals↗
PubMed1995

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Animals↗
PubMed1995

Co-operative regulation of endocytosis by three Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

Amino Acid Sequence↗
PubMed1995

Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells.

We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.

Animals↗
PubMed1995

Isolation of a murine cDNA clone encoding Rab19, a novel tissue-specific small GTPase.

Using a rapid amplification of cDNA ends (RACE) cloning approach, we have isolated a cDNA clone encoding Rab19, a novel small GTPase of the Rab subfamily contained within partial sequences previously described [Chavrier et al., Gene 112 (1992) 261-264]. Northern blot analysis of the distribution of the rab19 mRNA in various adult mouse tissues and NIH 3T3 fibroblasts revealed that rab19 is expressed in a tissue-specific manner. The rab19 transcript was detected at high levels in intestine, lung and spleen, and at a lower level in kidney. In contrast, liver, brain, heart and NIH 3T3 fibroblasts contain only very little or no detectable rab19 mRNA. Therefore, Rab19 is likely to represent a novel tissue- or cell type-specific small GTPase.

Amino Acid Sequence↗
PubMed1995

Prohibitin, an antiproliferative protein, is localized to mitochondria.

Prohibitin is a ubiquitously expressed protein with antiproliferative properties. When rat prohibitin tagged with a carboxy-terminal c-Myc epitope was expressed in baby hamster kidney cells the protein was targeted to mitochondria. In immunofluorescence microscopy prohibitin colocalized with a mitochondrial marker E3. Immunoelectron microscopy revealed that prohibitin was associated with the periphery of mitochondria. The amino-terminus of prohibitin shares characteristics of the known mitochondrial import signals, and positioning of the tag at the N-terminus causes accumulation of the protein in the cytoplasm. These findings help to direct functional studies on prohibitin and suggest that a mitochondrial protein may act as a tumor suppressor.

Amino Acid Sequence↗