Imaging intracellular membrane traffic and organelle dynamics using confocal microscopy

1991 ◽  
Vol 49 ◽  
pp. 390-391
Author(s):  
M. S. Cooper
Keyword(s):  
Confocal Microscopy ◽  
Golgi Apparatus ◽  
Optical Memory ◽  
Living Cells ◽  
Scanning Laser ◽  
Dynamic Interactions ◽  
Vesicular Traffic ◽  
Hippocampal Astrocytes ◽  
Laser Confocal Microscope ◽  
Endocytic Vesicles

Movements of exocytic and endocytic vesicles within cells are controlled in part by active transport along microtubules. The forces generated by microtubule-based transport are also known to strongly influence the distribution and shapes of membranous organelles, such as mitochondria and the endoplasmic reticulum. The ability to observe such dynamic interactions between cytoskeletal elements and fluorescently-labelled intracellular membranes in living cells has been improved recently by the advent of scanning laser confocal microscopy. Here we describe the use of this technique to study activities of the Golgi apparatus in living cells.Vesicular traffic, diffusive transport and morphological dynamics within the Golgi apparatus of cultured rat hippocampal astrocytes were imaged with a Bio-Rad MRC-500 scanning laser confocal microscope. Golgi elements were labelled with NBD-ceramide, a molecule which serves as a vital stain for the trans-most cisternae of Golgi stacks. Single scans of the specimen, obtained with the laser microscope's slow scan rate (4 sec/image), were stored sequentially on an optical memory disk recorder.

Identification and localization of Giα3 in the clonal adipocyte cell lines HGFu and Obl7

1993 ◽  
Vol 71 (11-12) ◽  
pp. 518-521 ◽  
Author(s):  
Monique Cadrin ◽  
Norma McFarlane-Anderson ◽  
Nicole Bégin-Heick
Keyword(s):  
Adipose Tissue ◽  
Confocal Microscopy ◽  
Golgi Apparatus ◽  
Subcellular Localization ◽  
G Protein ◽  
Cell Lines ◽  
Vesicular Traffic ◽  
Golgi System ◽  
Adipocyte Cell ◽  
Α3 Subunit

The HGFu and Ob17 cell lines, derived from adipose tissue of lean (+/?) and ob/ob mice, respectively, express several G-protein peptides. Investigation of the expression and subcellular localization of the Giα3 subunit showed that this peptide is associated with the Golgi apparatus. These findings indicate a role for this subunit in vesicular traffic and are in agreement with the view of the adipocyte as a secretory cell.Key words: preadipocytes, differentiation, immunofluorescence, confocal microscopy, obesity, Golgi system.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy

1991 ◽  
Vol 100 (4) ◽  
pp. 747-752 ◽  
Author(s):  
S. Chandra ◽  
E.P. Kable ◽  
G.H. Morrison ◽  
W.W. Webb
Keyword(s):  
Confocal Microscopy ◽  
Golgi Apparatus ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Ionophore A23187 ◽  
Elemental Distribution ◽  
Total Calcium ◽  
Potassium Sodium ◽  
Scanning Confocal Microscopy ◽  
Ion Microscopy

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-ceramide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PK1 porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 micron). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PK1 cells, about 2.5% in 3T3 cells and L5 cells.

Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 319-328
Author(s):  
F. Giorgi ◽  
P. Lucchesi ◽  
A. Morelli ◽  
M. Bownes
Keyword(s):  
Golgi Apparatus ◽  
Juvenile Hormone ◽  
Developmental Stages ◽  
Ovarian Follicles ◽  
Endocytic Pathway ◽  
Intermediate Cell ◽  
Wild Type ◽  
Vesicular Traffic

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.

Detecting Early Apoptosis in Whole Epithelial Sheets with a Caspase 3 Substrate, Phiphilux and Annexes V Using Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 600-601
Author(s):  
Kathy K. H. Svoboda
Keyword(s):  
In Situ Hybridization ◽  
Confocal Microscopy ◽  
General Principle ◽  
Caspase 3 ◽  
Living Cells ◽  
Corneal Epithelial ◽  
Early Apoptosis ◽  
Epithelial Sheets ◽  
Conventional Light Microscope

Many reagents have been developed recently to label living cells with substrates that will become fluorescent if an enzyme is active. The general principle is that the substrate will be taken up by living cells then detected only if the enzyme is active. These substrates work well with isolated individual cells, however, more difficulty can be encountered when studying whole tissues. Problems can range from substrate penetration into whole tissues to being able to detect the label effectively. We have used the chicken corneal epithelia for many studies, but the tissue is to thick to view with a conventional light microscope, therefore we have developed techniques using laser confocal microscopes to view this tissue in with a variety of techniques including in situ hybridization, immunohistochemistry, and vital dyes/stains.Whole embryonic corneal epithelial sheets can be isolated without the basal lamina.

scholarly journals Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy.

1990 ◽  
Vol 38 (3) ◽  
pp. 315-318 ◽  
Author(s):  
J M Robinson ◽  
B E Batten
Keyword(s):  
Hydrogen Peroxide ◽  
Confocal Microscopy ◽  
Scanning Laser ◽  
Reaction Products ◽  
Laser Confocal Microscopy ◽  
Light Microscope Level ◽  
New Approach ◽  
Detection Systems ◽  
Laser Reflectance ◽  
Hydrogen Peroxide Release

Scanning laser confocal microscopy was utilized to visualize sites of hydrogen peroxide release from stimulated neutrophils and lysosomal acid phosphatase in these and other cells using cerium in the detection systems. Imaging of the cerium-containing reactions was achieved by employing the reflectance mode of this instrument. Localization of these products at the light microscope level was direct and did not require other reactions to generate a visible product. This new approach to cerium cytochemistry should prove useful for many applications.

Confocal Microscopy of Mitochondrial Function in Living Cells

2006 ◽  
pp. 21-49
Author(s):  
John J. Lemasters ◽  
Ting Qian ◽  
Donna R. Trollinger ◽  
Wayne E. Cascio ◽  
Hisayuki Ohata ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Mitochondrial Function ◽  
Living Cells

Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles

2007 ◽  
Vol 293 (6) ◽  
pp. G1333-G1338 ◽  
Author(s):  
Svetlana G. Voronina ◽  
Mark W. Sherwood ◽  
Oleg V. Gerasimenko ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Acinar Cells ◽  
Cell Types ◽  
Living Cells ◽  
Pancreatic Acinar Cells ◽  
Extracellular Solution ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Cellular Organelles

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

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