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R G Parton

Publications and source records attributed to R G Parton.

At least 19 recordsLinked to original sources
PubMed1997

Caveolin-3 associates with developing T-tubules during muscle differentiation.

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the alpha 1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3-labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.

Amino Acid Sequence↗
PubMed1997

Major histocompatibility complex class I molecules mediate association of SV40 with caveolae.

Simian virus 40 (SV40) has been shown to enter mammalian cells via uncoated plasma membrane invaginations. Viral particles subsequently appear within the endoplasmic reticulum. In the present study, we have examined the surface binding and internalization of SV40 by immunoelectron microscopy. We show that SV40 associates with surface pits which have the characteristics of caveolae and are labeled with antibodies to the caveolar marker protein, caveolin-1. SV40 is believed to use major histocompatibility complex (MHC) class I molecules as cell surface receptors. Using a number of MHC class I-specific monoclonal antibodies, we found that both viral infection and association of virus with caveolae were strongly reduced by preincubation with anti-MHC class I antibodies. Because binding of SV40 to MHC class I molecules may induce clustering, we investigated whether antibody cross-linked class I molecules also redistributed to caveolae. Clusters of MHC class I molecules were indeed shown to be specifically associated with caveolin-labeled surface pits. Taken together, the results suggest that SV40 may make use of MHC class I molecule clustering and the caveolae pathway to enter mammalian cells.

Animals↗
PubMed1997

HSV infection of polarized epithelial cells on filter supports: implications for transport assays and protein localization.

Epithelial cell lines can be grown on filter supports and form polarized monolayers with distinct basolateral and apical plasma membrane domains. This property has been extensively used in cell biology to investigate epithelial cell function. To date, a major limitation of this approach has been the difficulty of obtaining transient gene expression in polarized epithelia. Here we present an approach to overcome this problem using gene transfer into polarized epithelial cells grown on filters using a herpes virus-based vector. Recombinant genes are inserted into a defective HSV-1 plasmid and packaged with a replication-incompetent HSV-1 helper virus into virus particles which are used to infect the polarized epithelial cells grown on filters. The transepithelial resistance of the cells is not affected by the addition of virus, and there are no detectable cytopathic effects.

Animals↗
PubMed1996

Endosome dynamics regulated by a Rho protein.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Actins↗
PubMed1996

And still they are moving.... dynamic properties of caveolae.

Caveolae are structures found on the surface of many mammalian cells. In the last few years the biogenesis and the function of these organelles have been intensively investigated but many challenging questions remain. One of these is whether caveolae are statically attached to the cytoplasmic surface of the plasma membrane or are moving to other intracellular organelles. Also the cycling of the caveolar coat component, VIP21-caveolin, is a subject of intensive discussion. The solution to these problems could give an insight into the understanding of caveolar function.

Animals↗
PubMed1996

M-caveolin, a muscle-specific caveolin-related protein.

Caveolae, small invaginations of the plasma membrane, are a characteristic feature of many mammalian cells. The best-characterised caveolar protein is the integral membrane protein, VIP21-caveolin. We now describe a novel homologue of VIP21-caveolin, M-caveolin, which is expressed exclusively in muscle. M-caveolin was shown to be expressed in differentiated myotubes but not myoblasts. Epitope-tagged M-caveolin expressed in non-muscle cells was targetted to surface caveolae where it colocalized with endogenous VIP21-caveolin. M-caveolin may play a specialised role in the caveolae of muscle cells.

Amino Acid Sequence↗
PubMed1996

Caveolae and caveolins.

In the past year, we have witnessed considerable progress towards an understanding of the workings of caveolae. Highlights include the identification of new caveolin family members, the characterization of VIP21-caveolin as a cholesterol-binding oligomeric protein, and evidence for functional interactions between caveolins and heterotrimeric G proteins. In addition, novel systems for caveolae purification and for studying caveolae biogenesis are starting to reveal insights into the molecular basis of caveolae formation and function.

Biological Transport↗
PubMed1996

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

Animals↗
PubMed1996

Rab11 regulates recycling through the pericentriolar recycling endosome.

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

Animals↗
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

The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.

Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.

Amino Acid Sequence↗
PubMed1995

M-caveolin, a muscle-specific caveolin-related protein.

Caveolae, small invaginations of the plasma membrane, are a characteristic feature of many mammalian cells. The best-characterised caveolar protein is the integral membrane protein, VIP21-caveolin. We now describe a novel homologue of VIP21-caveolin, M-caveolin, which is expressed exclusively in muscle. M-caveolin was shown to be expressed in differentiated myotubes but not myoblasts. Epitope-tagged M-caveolin expressed in non-muscle cells was targetted to surface caveolae where it colocalized with endogenous VIP21-caveolin. M-caveolin may play a specialised role in the caveolae of muscle cells.

Amino Acid Sequence↗
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

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

3T3 Cells↗
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↗
PubMed1995

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.

Amino Acid Sequence↗
PubMed1995

The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons.

The boundaries of the organelles of the biosynthetic endomembrane system are still controversial. In this paper we take advantage of the unique architectural organization of neurons to investigate the localization of a spectrum of compartment-specific markers with the goal of defining the location of the rough endoplasmic reticulum (ER), smooth ER, intermediate compartment, and the Golgi complex. Markers of the rough ER (signal sequence receptor), Golgi complex (mannosidase II), and the trans Golgi network (TGN38) were essentially restricted to the cell body and the initial segment of one of the cell's dendrites. In contrast the cytochemical reaction product for glucose 6 phosphate, a classical ER marker, in addition to staining ER structures in the cell body also reacted with smooth ER elements that extended into both axons and dendrites. These peripheral smooth ER elements also reacted at the immunofluorescence level for ER marker 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as for calnexin and protein disulfide isomerase. We also analyzed the location of rab1, rab2, p58, the KDEL receptor, and beta-subunit of coatomer. These intermediate compartment markers were found predominantly in the cell body but also extended to the proximal parts of the dendrites. Collectively, our data argue that the ER of hippocampal neurons consists of functionally and spatially distinct and separated domains, and they stress the power of the hippocampal neuron system for investigations of the organization of the ER by light microscopy.

Animals↗