High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells

Falk, Matthias M.; Lauf, Undine

doi:10.1002/1097-0029(20010201)52:3<251::aid-jemt1011>3.0.co;2-#

Cited by 47 publications

(6 citation statements)

References 58 publications

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“…Similar junction proteins from the arthropod Nephrops norvegicus, known as ''ductins'', have been described by atomic force microscopy as particles with an hexagonal lattice and a central pore-like depression (John et al 1997). Since these parasitic worms are acoelomic and their tissues organized as syncytia, we speculate that the numerous gapjunctions observed in adult T. solium tissues may be involved not only in the diffusion of ions, amino acids and secondary messengers, (Simon and Goodenough 1998;Musil et al 2000;Falk and Lauf 2001) but also in the transport of small molecules such as glucose (Sato et al 2002). These protein channels are inserted into membrane bilayers and appear as hexagonal structures with a central pore when viewed in cross-section.…”

Section: Discussionsupporting

confidence: 55%

“…Ultrastructural analysis of these junctions at high magnification led to their characterization as gap junctions due to their similarity to the junctions described in cestodes (Lumsden and Hildreth 1983;Richards and Arme 1983;Webb 1987;Conn 1988) and in mammalian tissue: increased electron density, a width of 13-18 nm in cross-section and an inner structure of discrete blocks of electron-dense material alternating with hypodense channels, some of which appeared as 2-3 nm ''open channels''. The junction proteins from vertebrates, known as ''connexins'', have been studied by various sophisticated methods and are formed by the assembly of two hemi-channels, termed connexons, which pair in adjoining plasma membranes to form the gap junction (Falk and Lauf 2001;Lal and Lin 2001) . The junction proteins from vertebrates, known as ''connexins'', have been studied by various sophisticated methods and are formed by the assembly of two hemi-channels, termed connexons, which pair in adjoining plasma membranes to form the gap junction (Falk and Lauf 2001;Lal and Lin 2001) .…”

Section: Discussionmentioning

confidence: 99%

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Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters

2003

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Taenia solium adults were grown in hamsters infected by feeding them with cysticerci from pig carcasses. Viable strobilae were collected from the hamster duodenum 20-60 days post-infection, fixed and processed for transmission electron microscopy (TEM). Fourteen strobilae were cut into pieces and embedded in individual blocks. Sections, stained with toluidine blue, were then photographed by light microscopy. Over 1,200 TEM images were obtained from selected blocks. Maturing proglottids exhibited a dense myofilament lattice of connecting fibers, each contained in sarcoplamsic extensions of myocytons and emitting cytoplasmic processes loosely attached to other cells, structures characterized as myocyton-myofilament-pseudopod units, which are interpreted as structures involved in the transport of cells and membrane-bound-glycogen from the germinative tissues to mature proglottids. Densely packed membrane-bound glycogen particles were found between the tegumentary cytons of the neck tissue, and as single-stranded particles between the tegumentary cytons of mature proglottids. These were wrapped around cell bodies in the parenchyma of maturing proglottids and as thin cytoplasmic strands between the testicular lobules of mature proglottids. A large number of cell-to-cell adhesions were identified as gap junctions connected to glycogen strands. We suggest that these are involved in the transport of glucose to differentiating tissues.

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters

2003

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“…The goal of the study reported here was to determine whether the Ca 2ϩ /CaMdependent inhibition of lens cell-to-cell communication was mediated in part by Cx43. This was accomplished with Cx43-transfected HeLa cells because these cells have been shown to provide an excellent system to examine the properties of homotypic gap junctions (17,18). Our results support the thesis that the Ca 2ϩ -dependent inhibition of Cx43 gap junctions results from the interaction of CaM with a cytoplasmic portion of Cx43 that excludes the bulk of its COOH-terminal region.…”

supporting

confidence: 74%

Intracellular calcium regulation of connexin43

Lurtz

Louis²

2007

American Journal of Physiology-Cell Physiology

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The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.

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“…Specific proteins can now be tagged with florescent proteins (FPs) that fluoresce in blue, green, yellow, orange or red region10. In conjunction with basic light microscopy, many molecular interactions can be implied based on simple colocalization studies 11. This colocalization approach is however limited by the resolution of light microscopy (typically 0.2 μm).…”

Section: Introductionmentioning

confidence: 99%

Photophysical properties of Cerulean and Venus fluorescent proteins

et al. 2009

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Cerulean and Venus are recently developed fluorescent proteins, often used as a donor-acceptor pair by researchers in FRET based co-localization studies. We characterized the fluorescent properties of these two proteins in a broad spectral range (form Ultra-Violet to Visible region). Excitation spectra, lifetimes and polarization spectra show significant energy transfer from aromatic amino acids to the fluorescent protein chromophore. High steady-state anisotropy values and the lack of a fast component in anisotropy decays show that the fluorescent protein chromophore is rigidly fixed within the protein structure. Furthermore, we show that the chromophores are not accessible to external quenchers such as acrylamide or potassium iodide (KI), allowing the removal of 'unwanted' background in the environment with external quencher, while leaving the Cerulean/Venus fluorescence unchanged.

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High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells

Cited by 47 publications

References 58 publications

Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters

Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters

Intracellular calcium regulation of connexin43

Photophysical properties of Cerulean and Venus fluorescent proteins

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