Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking

2007 ◽  
Vol 293 (1) ◽  
pp. G165-G177 ◽  
Author(s):  
Jack A. Valentijn ◽  
Laura van Weeren ◽  
Anton Ultee ◽  
Abraham J. Koster
Keyword(s):  
Laser Scanning ◽  
Secretory Granules ◽  
Physiological Role ◽  
Acinar Cells ◽  
Goblet Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Unexpected Finding ◽  
Brunner’S Gland ◽  
Brunner's Gland

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, β-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.

scholarly journals Carbonic anhydrase isoenzyme II is located in corticotrophs of the human pituitary gland.

1996 ◽  
Vol 44 (3) ◽  
pp. 245-250 ◽  
Author(s):  
A K Parkkila ◽  
S Parkkila ◽  
H Rajaniemi
Keyword(s):  
Carbonic Anhydrase ◽  
Pituitary Gland ◽  
Laser Scanning ◽  
Physiological Role ◽  
Laser Scanning Microscopy ◽  
Immunofluorescence Staining ◽  
Confocal Laser ◽  
Double Immunofluorescence ◽  
Human Pituitary ◽  
Human Pituitary Gland

We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.

scholarly journals Cyclic AMP accelerates calcium waves in pancreatic acinar cells

2008 ◽  
Vol 294 (6) ◽  
pp. G1328-G1334 ◽  
Author(s):  
Ahsan U. Shah ◽  
Wayne M. Grant ◽  
Sahibzada U. Latif ◽  
Zahir M. Mannan ◽  
Alexander J. Park ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Wave Speed ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Laser Scanning Microscopy ◽  
Pancreatic Acinar Cells ◽  
Confocal Laser ◽  
Channel Opening ◽  
Dependent Protein

Cytosolic Ca2+ (Ca[Formula: see text]) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca2+ wave generated by the Ca2+-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca2+ channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 μM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca[Formula: see text] was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 μM)-stimulated Ca2+ wave was 8.63 ± 0.68 μm/s; it increased to 19.66 ± 2.22 μm/s (* P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 μM). The Ca2+ rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca2+ elevation were not significantly affected by cAMP. The effect of cAMP on Ca2+ waves also did not appear to depend on extracellular Ca2+. However, the ryanodine receptor (RyR) inhibitor dantrolene (100 μM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 μM). 8-(4-chloro-phenylthio)-2′- O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca2+ wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.

Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy

2002 ◽  
Vol 363 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Mica OHARA-IMAIZUMI ◽  
Yoko NAKAMICHI ◽  
Toshiaki TANAKA ◽  
Hidenori KATSUTA ◽  
Hitoshi ISHIDA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Ph Sensitive ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Min6 Cells ◽  
Green Fluorescent

The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic β-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH7.4, but not at approximately pH5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin—pHluorin, time-lapse confocal laser scanning microscopy (5 or 10s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50mM KCl and 22mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin—pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽  
2010 ◽  
Vol 9 (10) ◽  
pp. 7-15
Author(s):  
HANNA KOIVULA ◽  
DOUGLAS BOUSFIELD ◽  
MARTTI TOIVAKKA
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Print Quality ◽  
Length Scale ◽  
Offset Printing ◽  
Significant Difference ◽  
Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

ACTIVATED SLUDGE FLOC CHARACTERIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

2012 ◽  
Vol 11 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Szabolcs Szilveszter ◽  
Botond Raduly ◽  
Szilard Bucs ◽  
Beata Abraham ◽  
Szabolcs Lanyi ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser
Sign in / Sign up

Export Citation Format

Share Document