scholarly journals Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

2006 ◽  
Vol 100 (5) ◽  
pp. 1340-1350 ◽  
Author(s):  
Shin-Rong Hwang ◽  
Audrey O’Neill ◽  
Steven Bark ◽  
Thierry Foulon ◽  
Vivian Hook
Keyword(s):  
Subcellular Localization ◽  
Molecular Identification ◽  
Secretory Vesicle ◽  
Chromaffin Granules

scholarly journals Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

1994 ◽  
Vol 299 (2) ◽  
pp. 473-479 ◽  
Author(s):  
H Sengeløv ◽  
F Boulay ◽  
L Kjeldsen ◽  
N Borregaard
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Plasma Membranes ◽  
Secretory Vesicle ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Beta Band ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Specific Granules ◽  
Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

scholarly journals Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.

1996 ◽  
Vol 44 (12) ◽  
pp. 1481-1488 ◽  
Author(s):  
V A Tanner ◽  
T Ploug ◽  
J H Tao-Cheng
Keyword(s):  
Subcellular Localization ◽  
Pc12 Cells ◽  
Cell Cultures ◽  
Transmembrane Protein ◽  
Secretory Granules ◽  
Secretory Vesicle ◽  
Secretory Vesicles ◽  
Experimental Conditions ◽  
Gold Probe ◽  
20 Nm

We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures for cell cultures. The advantages and related caveats of this method are discussed. SV2 was distinctly localized on dusters of synaptic vesicles and large dense-cored vesicles (LDCV). The distribution of SV2 on these two types of secretory vesicles was compared quantitatively to that of another secretory vesicle-associated transmembrane protein, synaptophysin. In cultures under similar experimental conditions, the ratio of SV2 vs synaptophysin ICC staining on synaptic vesicle dusters was about 1:1, whereas it was about 9:1 on LDCV membranes. Furthermore, whereas SV2 is localized on the membranes of the LDCVs, chromogranin A, an acidic protein in secretory granules, is clearly in the core of the LDCVs. This is the first demonstration of these two antigens in such dose (approximately 20 nm) yet distinct compartments within a single organelle.

scholarly journals Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

1992 ◽  
Vol 116 (2) ◽  
pp. 359-365 ◽  
Author(s):  
D Scheuner ◽  
C D Logsdon ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Secretory Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Chromaffin Granules ◽  
Chromaffin Granule ◽  
Vesicle Membrane ◽  
Sequence Of Events ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Adrenal Chromaffin

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

Electron microscopy in diagnostic virology

1984 ◽  
Vol 42 ◽  
pp. 70-73
Author(s):  
S. E. Miller
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Molecular Identification ◽  
Point Of View ◽  
Negative Stain ◽  
Viral Diagnosis ◽  
Electron Microscopist ◽  
Diagnostic Virology

The techniques for detecting viruses are many and varied including FAT, ELISA, SPIRA, RPHA, SRH, TIA, ID, IEOP, GC (1); CF, CIE (2); Tzanck (3); EM, IEM (4); and molecular identification (5). This paper will deal with viral diagnosis by electron microscopy and will be organized from the point of view of the electron microscopist who is asked to look for an unknown agent--a consideration of the specimen and possible agents rather than from a virologist's view of comparing all the different viruses. The first step is to ascertain the specimen source and select the method of preparation, e. g. negative stain or embedment, and whether the sample should be precleared by centrifugation, concentrated, or inoculated into tissue culture. Also, knowing the type of specimen and patient symptoms will lend suggestions of possible agents and eliminate some viruses, e. g. Rotavirus will not be seen in brain, nor Rabies in stool, but preconceived notions should not prejudice the observer into missing an unlikely pathogen.

Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells

1990 ◽  
Vol 48 (3) ◽  
pp. 952-953
Author(s):  
Gemma A.J. Kuijpers ◽  
Harvey B. Pollard
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Chromaffin Cells ◽  
Cell Activation ◽  
Structural Information ◽  
Dependent Manner ◽  
Chromaffin Granules ◽  
Immuno Electron Microscopy ◽  
Ultrathin Sections ◽  
Adrenal Chromaffin

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+ upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+ (1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.

Investigating the functional link between TMEM165 and SPCA1

2019 ◽  
Vol 476 (21) ◽  
pp. 3281-3293 ◽  
Author(s):  
Elodie Lebredonchel ◽  
Marine Houdou ◽  
Hans-Heinrich Hoffmann ◽  
Kateryna Kondratska ◽  
Marie-Ange Krzewinski ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Complete Loss ◽  
Protein Family ◽  
Uncharacterized Protein ◽  
Functional Link ◽  
P Type

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

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