scholarly journals Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

2010 ◽  
Vol 298 (5) ◽  
pp. L626-L636 ◽  
Author(s):  
Heike Schlenz ◽  
Wolfgang Kummer ◽  
Gitte Jositsch ◽  
Jürgen Wess ◽  
Gabriela Krasteva
Keyword(s):  
Smooth Muscle ◽  
Signaling Pathways ◽  
Laser Scanning ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Caveolin 1

Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R−/− and M3R−/− mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

scholarly journals Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET)

2003 ◽  
Vol 31 (5) ◽  
pp. 1020-1027 ◽  
Author(s):  
D.S. Lidke ◽  
P. Nagy ◽  
B.G. Barisas ◽  
R. Heintzmann ◽  
J.N. Post ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Laser Scanning ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

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.

scholarly journals Influence of Aging on Corrosion Behaviour of the 6061 Cast Aluminium Alloy

Materials ◽  
2021 ◽  
Vol 14 (8) ◽  
pp. 1821
Author(s):  
Ting He ◽  
Wei Shi ◽  
Song Xiang ◽  
Chaowen Huang ◽  
Ronald G. Ballinger
Keyword(s):  
Aluminium Alloy ◽  
Laser Scanning ◽  
Corrosion Behaviour ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Kelvin Probe ◽  
Force Microscopy ◽  
The Matrix ◽  
6061 Aluminium Alloy

The influence of AlFeSi and Mg2Si phases on corrosion behaviour of the cast 6061 aluminium alloy was investigated. Scanning Kelvin probe force microscopy (SKPFM), electron probe microanalysis (EPMA), and in situ observations by confocal laser scanning microscopy (CLSM) were used. It was found that Mg2Si phases were anodic relative to the matrix and dissolved preferentially without significantly affecting corrosion propagation. The AlFeSi phases’ influence on 6061 aluminium alloy local corrosion was greater than that of the Mg2Si phases. The corroded region width reached five times that of the AlFeSi phase, and the accelerating effect was terminated as the AlFeSi dissolved.

scholarly journals In Situ 3D-Imaging of the Inner Ear Synapses with a Cochlear Implant

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 301
Author(s):  
Kathrin Malfeld ◽  
Nina Armbrecht ◽  
Holger A. Volk ◽  
Thomas Lenarz ◽  
Verena Scheper
Keyword(s):  
Cochlear Implant ◽  
Inner Ear ◽  
Laser Scanning ◽  
3D Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Sensory Cells ◽  
Residual Hearing ◽  
Preparation Methods

In recent years sensorineural hearing loss was found to affect not exclusively, nor at first, the sensory cells of the inner ear. The sensory cells’ synapses and subsequent neurites are initially damaged. Auditory synaptopathies also play an important role in cochlear implant (CI) care, as they can lead to a loss of physiological hearing in patients with residual hearing. These auditory synaptopathies and in general the cascades of hearing pathologies have been in the focus of research in recent years with the aim to develop more targeted and individually tailored therapeutics. In the current study, a method to examine implanted inner ears of guinea pigs was developed to examine the synapse level. For this purpose, the cochlea is made transparent and scanned with the implant in situ using confocal laser scanning microscopy. Three different preparation methods were compared to enable both an overview image of the cochlea for assessing the CI position and images of the synapses on the same specimen. The best results were achieved by dissection of the bony capsule of the cochlea.

Altered Cytoskeleton in Smooth Muscle of Aganglionic Bowel

2002 ◽  
Vol 126 (6) ◽  
pp. 692-696
Author(s):  
Laszlo Nemeth ◽  
Udo Rolle ◽  
Prem Puri
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Laser Scanning ◽  
Hirschsprung Disease ◽  
Muscle Cells ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Normal Bowel ◽  
Pull Through

Abstract Context.—Intestinal motility is under the control of smooth muscle cells, enteric plexus, and hormonal factors. In Hirschsprung disease (HD), the aganglionic colon remains spastic or tonically enhanced and unable to relax. The smooth muscle cell's cytoskeleton consists of proteins or structures whose primary function is to link or connect protein filaments to each other or to the anchoring sites. Dystrophin is a subsarcolemmal protein with a double adhesion property, one between the membrane elements and the contractile filaments of the cytoskeleton and the other between the cytoskeletal proteins and the extracellular matrix. Desmin and vinculin are functionally related proteins that are present in the membrane-associated dense bodies in the sarcolemma of the smooth muscle cells. Objective.—To examine the distribution of the cytoskeletal proteins in the smooth muscle of the aganglionic bowel. Design.—Bowel specimens from ganglionic and aganglionic sections of the colon were collected at the time of pull-through surgery from 8 patients with HD. Colon specimens collected from 4 patients at the time of bladder augmentation acted as controls. Anti-dystrophin, anti-desmin, and anti-vinculin antibodies were used for fluorescein immunostaining using confocal laser scanning microscopy. Results.—Moderate to strong dystrophin immunoreactivity was observed at the periphery of smooth muscle fibers in normal bowel and ganglionic bowel from patients with HD, whereas dystrophin immunoreactivity was either absent or weak in the smooth muscle of aganglionic colon. Moderate to strong cytoplasmic immunostaining for vinculin and desmin was seen in the smooth muscle of normal bowel and ganglionic bowel from patients with HD, whereas vinculin and desmin staining in the aganglionic colon was absent or weak. Conclusion.—This study demonstrates that the cytoskeletal proteins are abundant in the smooth muscle of normal bowel, but are absent or markedly reduced in the aganglionic bowel of HD. As cytoskeletal proteins are required for the coordinated contraction of muscle cells, their absence may be responsible for the motility dysfunction in the aganglionic segment.

scholarly journals In Situ Characterization ofNitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

2001 ◽  
Vol 67 (11) ◽  
pp. 5273-5284 ◽  
Author(s):  
Holger Daims ◽  
Jeppe L. Nielsen ◽  
Per H. Nielsen ◽  
Karl-Heinz Schleifer ◽  
Michael Wagner
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
Laser Scanning ◽  
Wastewater Treatment Plants ◽  
Carbon Sources ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rrna Gene ◽  
Phylogenetic Affiliation

ABSTRACT Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of theNitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genusNitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates ofNitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospiramicrocolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources byNitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, theNitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3 − or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by theNitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

Monitoring the development of anaerobic biofilms using fluorescent in situ hybridization and confocal laser scanning microscopy

2000 ◽  
Vol 41 (12) ◽  
pp. 69-77 ◽  
Author(s):  
J. C. Araujo ◽  
G. Brucha ◽  
J. R. Campos ◽  
R. F. Vazoller
Keyword(s):  
In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Fluorescent In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Anaerobic Consortium

In this study we investigated the development of anaerobic biofilm using a laboratory reactor. We were especially interested in comparing the organization of anaerobic cells (particularly those that are very common in domestic sewage sludge) in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescent in situ hybridization (FISH) with domain and group specific probes directed against 16S ribosomal RNA were used to quantify microbial composition in the biofilm. FISH and confocal laser scanning microscopy (CLSM) were used to elucidate spatial distribution of microbes in the biofilms. Two experiments were carried out, one with pure methanogenic organisms and the other with a microbial anaerobic consortium. The pure methanogen cultures, Methanobacterium formicicum (DSM 1535); Methanosaeta concilli (DSM 3671) and Methanosarcina barkeri (DSM 800) were used to seed the modified Robbins Device (MRD) to allow the development of biofilms on polypropylene and glass surfaces during the 9-days experiment. The results showed that all the three species were colonizing both surfaces after two and nine days of experimental period. In another experiment, with polypropylene coupons only, MRD was seeded with a microbial anaerobic consortium and biofilm formation was studied during 11 days. At the end of this period, the biofilms generated were of uneven thickness with areas of minimal or no surface coverage and areas where the biofilm attained a thickness of 7.0 to 9.0 μm as revealed by CLSM. The results showed that the modified Robbins Device together with the fluorescent in situ hybridization and confocal laser scanning microscopy are suitable tools to study anaerobic biofilm development in different kinds of support materials.

Sign in / Sign up

Export Citation Format

Share Document