Biomedical subjects

R G Parton

Publications and source records attributed to R G Parton.

At least 73 records · Page 4Linked to original sources

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↗

PubMed1995

VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.

Animals↗

PubMed1995

Rapid processing of filter-grown cells for Epon embedding.

A simple and rapid method for fixation, processing, and Epon embedding of cells grown on a filter support is described. The results show that it is possible to embed filter-grown cells in resin within 30 min of applying the primary fixative and still to achieve good ultrastructural preservation.

Animals↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis.

Small GTPases of the rab family control distinct steps of intracellular transport. The function of their GTPase activity is not completely understood. To investigate the role of the nucleotide state of rab5 in the early endocytic pathway, the effects of two mutants with opposing biochemical properties were tested. The Q79L mutant of rab5, analogous with the activating Q61L mutant of p21-ras, was found to have a strongly decreased intrinsic GTPase activity and was, unlike wild-type rab5, found mainly in the GTP-bound form in vivo. Expression of this protein in BHK and HeLa cells led to a dramatic change in cell morphology, with the appearance of unusually large early endocytic structures, considerably larger than those formed upon overexpression of wild-type rab5. An increased rate of transferrin internalization was observed in these cells, whereas recycling was inhibited. Cytosol containing rab5 Q79L stimulated homotypic early endosome fusion in vitro, even though it contained only a small amount of the isoprenylated protein. A different mutant, rab5 S34N, was found, like the inhibitory p21-ras S17N mutant, to have a preferential affinity for GDP. Overexpression of rab5 S34N induced the accumulation of very small endocytic profile and inhibited transferrin endocytosis. This protein inhibited fusion between early endosomes in vitro. The opposite effects of the rab5 Q79L and S34N mutants suggest that rab5:GTP is required prior to membrane fusion, whereas GTP hydrolysis by rab5 occurs after membrane fusion and functions to inactivate the protein.

Animals↗

PubMed1994

The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

Animals↗

PubMed1994

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus.

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.

Animals↗

PubMed1994

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↗

PubMed1994

Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae.

The ultrastructural distribution of the ganglioside GM1 was investigated in A431 cells. After fixation, the cells were frozen in liquid nitrogen, freeze-substituted, and then embedded in Lowicryl resin at -45 degrees C. By use of the cholera toxin-binding subunit adsorbed to gold as a specific probe to label on the sections, GM1 was shown to be present in endocytic organelles, in the trans-Golgi network, and on the plasma membrane, but was not detectable in the endoplasmic reticulum. GM1 was not distributed uniformly over the plasma membrane but was concentrated approximately four-fold in non-coated invaginations. These were identified as caveolae by labeling frozen sections of cholera toxin-gold surface-labeled cells with antibodies to VIP-21/caveolin. The results strengthen the functional analogy between caveolae and sorting domains of the TGN in polarized epithelial cells.

Carrier Proteins↗

PubMed1994

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↗

PubMed1994

Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex.

Among the small GTPases of the Ras family, Rap proteins exhibit the highest homology with p21Ras. The four Rap proteins so far identified constitute two subgroups, comprising the Rap1(A,B) and the Rap2(A,B) proteins. The intracellular location of Rap1A, Rap1B and Rap2A proteins was investigated in mammalian cells by confocal immunofluorescence microscopy. Using a specific anti-Rap1 affinity-purified antibody, both Rap1A and Rap1B proteins were localized to late endocytic compartments (late endosomes/lysosomes) in fibroblasts. The localization of the Rap1A and B proteins transiently overexpressed with the vaccinia T7 system was identical to that observed for endogenous Rap1 proteins. In contrast, epitope-tagged Rap2A protein colocalized with several markers of the Golgi complex, thus indicating that its site of function was distinct from that of Rap1A. In addition, morphological and subcellular fractionation studies provided evidence for the association of Rap1 proteins with phagosomes displaying biochemical features of late endocytic structures in J774 macrophages. Thus, the localization of Rap1A and Rap1B implicates their involvement in late endocytic/phagocytic processes.

Animals↗

PubMed1993

Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells.

A specific polyclonal antibody was used to investigate the subcellular distribution of the small GTPase, rab11p, in the neuroendocrine cell line, PC12. We took advantage of a previously described pulse-chase protocol based on sulfation to examine the distribution of rab11 along the secretory pathway. Using the rab11 antiserum, but not serum depleted of rab11 antibodies, we were able to specifically immunoisolate markers of the constitutive and the regulated secretory pathway in the trans-Golgi network (TGN) as well as after their exit from this compartment (constitutive secretory vesicles, immature, and mature secretory granules). We therefore conclude that rab11p is associated with the TGN and with TGN-derived vesicles of both the constitutive and the regulated secretory pathway in PC12 cells.

Animals↗

PubMed1993

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons.

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.

Journal Article↗

PubMed1993

Cell biology of neuronal endocytosis.

Endocytosis is the process by which cells take in fluid and components of the plasma membrane. In this way cells obtain nutrients and trophic factors, retrieve membrane proteins for degradation, and sample their environment. In neuronal cells endocytosis is essential for the recycling of membrane after neurotransmitter release and plays a critical role during early developmental stages. Moreover, alterations of the endocytic pathway have been attributed a crucial role in the pathophysiology of certain neurological diseases. Although well characterized at the ultrastructural level, little is known of the dynamics and molecular organization of the neuronal endocytic pathways. In this respect most of our knowledge comes from studies of non-neuronal cells. In this review we will examine the endocytic pathways in neurons from a cell biological viewpoint by making comparisons with non-neuronal cells and in particular with another polarized cell, the epithelial cell.

Axonal Transport↗

PubMed1993

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.

Animals↗

PubMed1993

The immunofluorescent era of membrane traffic.

In recent years immunofluorescence microscopy has been increasingly used to study membrane traffic. In this article seven electron microscopists, all with considerable experience in using light microscopy, take a critical look at the immunofluorescence approach and argue that results obtained with this method are often overinterpreted.

Journal Article↗