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W B Huttner

Publications and source records attributed to W B Huttner.

At least 55 records · Page 3Linked to original sources
PubMed1997

Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

Animals↗
PubMed1997

Neuroepithelial cells downregulate their plasma membrane polarity prior to neural tube closure and neurogenesis.

Cell differentiation often involves changes in cell polarity. In this study we show that neuroepithelial cells, the progenitors of all neurons and macroglial cells of the vertebrate central nervous system, downregulate the polarized delivery to the apical and basolateral plasma membrane domains during development. Upon infection of the neuroepithelium of mouse embryos with fowl plague virus (FPV), polarized delivery of the viral envelope hemagglutinin, an apical marker, occurred at the neural plate stage (E8), but was downregulated at the open neural tube stage (E9). Upon infection with vesicular stomatitis virus, the viral envelope G protein, a basolateral marker, showed an unpolarized delivery not only at the open neural tube stage, but already at the neural plate stage. These results show that a progressive downregulation of plasma membrane polarity of neuroepithelial cells precedes neural tube closure and the onset of neurogenesis.

Animals↗
PubMed1997

Asymmetric division and polarity of neuroepithelial cells.

Neuroepithelial cells, the progenitors to the CNS neurons and glia, undergo both symmetric and asymmetric divisions. Symmetric divisions underlie the proliferation of neuroepithelial cells that predominates early in CNS development. Asymmetric divisions are thought to generate the cell types derived from neuroepithelial cells, such as neurons. Insight into the mechanism of asymmetric division of neuroepithelial cells has come from two lines of research, the study of their epithelial polarity and the analysis of the expression of vertebrate homologues of proteins known to be involved in cell fate determination in Drosophila.

Animals↗
PubMed1996

Loss of occludin and functional tight junctions, but not ZO-1, during neural tube closure--remodeling of the neuroepithelium prior to neurogenesis.

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.

Amnion↗
PubMed1996

Overall lack of regulated secretion in a PC12 variant cell clone.

A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and alpha-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-beta-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation.

Animals↗
PubMed1996

Expression of tyrosine-sulfated secretory proteins in Xenopus laevis oocytes. Differential export of constitutive and regulated proteins.

Xenopus laevis oocytes were used to study the tyrosine sulfation and secretion of exogenous proteins. Secretogranin II (SgII), a tyrosine-sulfated protein found in secretory granules of a wide variety of endocrine cells and neurons, became tyrosine-sulfated by the oocytes when expressed by injection of poly(A)-rich RNA isolated from the neuroendocrine cell line PC12. The same result was observed when SgII was expressed from cloned SgII cRNA, showing that its tyrosine sulfation did not require the coexpression of exogenous tyrosylprotein sulfotransferase (TPST) but occurred by means of the endogenous oocyte TPST. Sulfophilin, an artificial protein consisting of 12 repeats of a heptapeptide tyrosine-sulfation site, was highly sulfated upon injection of its RNA, indicating the presence of TPST levels sufficient for stoichiometric sulfation of appropriate reporter proteins. Comparison of the secretion of [35S]sulfate-labelled SgII with that of sulfophilin and an exogenous heparan sulfate proteoglycan (HSPG), two proteins delivered to the cell surface by the constitutive pathway of secretion, revealed striking differences. The majority of sulfophilin and the HSPG was found in the medium, whereas that of SgII was found intracellularly. Prolactin, another secretory granule protein, showed the same secretion behaviour as SgII. These results show that oocytes express TPST and that these cells secrete constitutive and regulated secretory proteins in a differential manner.

Animals↗
PubMed1996

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.

Adrenocorticotropic Hormone↗
PubMed1996

A role for ADP-ribosylation factor 1, but not COP I, in secretory vesicle biogenesis from the trans-Golgi network.

A synthetic N-myristoylated peptide corresponding to the amino-terminal domain of ADP-ribosylation factor 1 (ARF1) markedly increases, in a cell-free system using post-nuclear supernatant from PC12 cells, the biogenesis of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network (TGN). The related N-myristoylated ARF4 peptide only weakly stimulates, and the non-myristoylated ARF1 and ARF4 peptides inhibit, the biogenesis of these secretory vesicles. In a modified cell-free system using TGN membranes, coatomer-depleted cytosol supports the biogenesis of TGN-derived secretory vesicles to the same extent as control cytosol. These results suggest a role for ARF1, but not the COP I coat, in secretory vesicle biogenesis from the TGN, possibly via the activation of phospholipase D.

ADP-Ribosylation Factor 1↗
PubMed1996

Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian genomes.

Chromogranin A, chromogranin B, and secretogranin II, members of the chromogranin/secretogranin secretory protein family, are overexpressed in some human hereditary maladies and may have arisen, in part, from common ancestor genes. To understand better the mammalian chromosomal dispersion of this gene family and to facilitate studies of these genes in human illnesses and their animal models, we positioned the locus of each member in the rat, mouse, and human genomes. Our results indicate that each locus lies in a region of locally syntenic chromosomal homology across the three species.

Animals↗
PubMed1995

A role for phosphatidylinositol transfer protein in secretory vesicle formation.

Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and their fusion with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids. Here we report, using a previously established cell-free system derived from a neuroendocrine cell line, the purification of cytosolic factors that stimulate the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. One such factor, referred to as CAST1, was identified as the alpha and beta isoforms of the mammalian phosphatidylinositol transfer protein (PtdIns-TP) (refs 3-5). The yeast PtdIns-TP, SEC14p (ref. 6), which has no sequence homology to mammalian PtdIns-TP (refs 7,8), was able to substitute for the mammalian PtdIns-TP in secretory vesicle formation. Our results suggest a highly conserved role for phosphoinositides in vesicle formation.

Animals↗
PubMed1995

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Animals↗
PubMed1995

Synaptotagmin I- and II-deficient PC12 cells exhibit calcium-independent, depolarization-induced neurotransmitter release from synaptic-like microvesicles.

Synaptotagmin I- and II-deficient PC12 cells (Shoji-Kasai et al. [1]) were used to compare the role of this protein in the calcium-dependent exocytosis of secretory granules and synaptic-like microvesicles (SLMVs). While neither catecholamine nor protein secretion from secretory granules were altered, the depolarization-induced acetylcholine release from SLMVs was no longer calcium-dependent. We propose that within the exocytotic process of SLMVs, there exist two depolarization-induced steps. One is calcium-dependent and no longer present in synaptotagmin I- and II-deficient cells. The other is induced by depolarization, does not require calcium, and suffices to trigger neurotransmitter release from SLMVs in synaptotagmin I- and II-deficient PC12 cells.

Acetylcholine↗
PubMed1994

An elevation of cytosolic protein phosphorylation modulates trimeric G-protein regulation of secretory vesicle formation from the trans-Golgi network.

The role of protein phosphorylation in the formation of secretory vesicles from the trans-Golgi network (TGN) and in the regulation of this process by TGN-associated trimeric G-proteins was investigated, using a previously established and a novel cell-free system derived from the neuroendocrine cell line PC12. In the absence of exogenous activators of trimeric G-proteins, okadaic acid, an inhibitor of protein serine/threonine phosphatase types 1, 2A, and PPX, had no significant effect on secretory vesicle formation as reconstituted in a postnuclear supernatant. However, okadaic acid antagonized the inhibition of secretory vesicle formation which occurred upon activation of trimeric G-proteins by either aluminum fluoride or guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S). Microcystin-LR, a protein phosphatase inhibitor structurally distinct from okadaic acid, also antagonized the trimeric G-protein-mediated inhibition of secretory vesicle formation but, in contrast to okadaic acid, alone was sufficient to stimulate this process. The antagonistic effect of the phosphatase inhibitors was abolished by a broad spectrum protein kinase inhibitor, staurosporine, which alone, however, did not affect vesicle formation. The effect of okadaic acid was promoted by activators of protein kinase C (phorbol myristate acetate) and protein kinase A (cyclic AMP). To investigate the subcellular localization of the phosphoprotein that is involved in the antagonistic effect of protein phosphatase inhibitors, a novel cell-free system was established which reconstitutes the formation of secretory vesicles from TGN membranes supplemented with cytosol. Using this cell-free system, the relevant phosphoprotein was found to reside in the cytosol. In conclusion, our results suggest that serine/threonine protein phosphorylation is not required for secretory vesicle formation from the TGN but modulates, via a cytosolic phosphoprotein, the regulation of this process by TGN-associated trimeric G-proteins.

Alkaloids↗
PubMed1994

The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-Golgi network.

Chromogranin B (secretogranin I), a protein sorted to secretory granules in many endocrine cells and neurons, undergoes selective aggregation during the sorting process in the trans-Golgi network. Reduction of the single, highly conserved intramolecular disulfide bond of chromogranin B by exposure of intact PC12 cells to the thiol reducing agent dithiothreitol has previously been shown to cause its missorting to the constitutive pathway of secretion. Using saponin perforation of membrane vesicles in aggregative buffer mimicking the milieu in the lumen of the trans-Golgi network (pH 6.4, 10 mM calcium), we show here that treatment with dithiothreitol does not prevent the aggregation of chromogranin B in this compartment. This implies that the loop in the chromogranin B polypeptide that is formed by the disulfide bond has a critical role in the membrane recognition of aggregated chromogranin B during secretory granule formation.

Animals↗
PubMed1994

Protein tyrosine sulfation, 1993--an update.

Sulfation is the most abundant post-translational modification of tyrosine residues and occurs in many soluble and membrane proteins passing through the secretory pathway of metazoan cells. The sulfation reaction is catalysed by tyrosylprotein sulfotransferase, a membrane-bound enzyme of the trans-Golgi-network. Tyrosylprotein sulfotransferase has been purified and its substrate specificity characterized. Tyrosine sulfation has been shown to be important for protein-protein interactions occurring during the intracellular transport of proteins and upon their secretion.

Animals↗