The origin of annular junctions: a mechanism of gap junction internalization

2001 ◽  
Vol 114 (4) ◽  
pp. 763-773 ◽  
Author(s):  
K. Jordan ◽  
R. Chodock ◽  
A.R. Hand ◽  
D.W. Laird
Keyword(s):  
Cell Surface ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Time Lapse ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junction Channels ◽  
Normal Rat ◽  
Connexin Proteins ◽  
Green Fluorescent

Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3–6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.

scholarly journals 3′-Untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation

2000 ◽  
Vol 352 (1) ◽  
pp. 109-115 ◽  
Author(s):  
Carlo M. Di LIEGRO ◽  
Marianna BELLAFIORE ◽  
José M. IZQUIERDO ◽  
Anja RANTANEN ◽  
José M. CUEZVA
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
In Vitro Translation ◽  
Mitochondrial Transcription Factor ◽  
Normal Rat ◽  
Green Fluorescent

Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3´-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3´-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

scholarly journals Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase

2007 ◽  
Vol 408 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Maria A. Brehm ◽  
Tobias M. H. Schenk ◽  
Xuefei Zhou ◽  
Werner Fanick ◽  
Hongying Lin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Stress Granules ◽  
Site Directed Mutagenesis ◽  
Normal Rat ◽  
High Concentrations ◽  
Green Fluorescent ◽  
Sites Of Action

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)–IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

scholarly journals Gating Properties of Gap Junction Channels Assembled from Connexin43 and Connexin43 Fused with Green Fluorescent Protein

2001 ◽  
Vol 81 (1) ◽  
pp. 137-152 ◽  
Author(s):  
Feliksas F. Bukauskas ◽  
Angele Bukauskiene ◽  
Michael V.L. Bennett ◽  
Vytas K. Verselis
Keyword(s):  
Green Fluorescent Protein ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Gap Junction Channels ◽  
Green Fluorescent

scholarly journals Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

2005 ◽  
Vol 16 (11) ◽  
pp. 5247-5257 ◽  
Author(s):  
Judy K. VanSlyke ◽  
Linda S. Musil
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Proteasome Inhibitors ◽  
Cell Surface Biotinylation ◽  
Normal Rat ◽  
And Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

Transport of Connexins and Gap Junction Turnover in Living Cells Visualized Using Green Fluorescent Protein Tagged Connexins.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1020-1021
Author(s):  
D.W. Laird ◽  
K. Jordan ◽  
P. Fistouris ◽  
J.L. Solan ◽  
P.D. Lampe ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Living Cells ◽  
Adjacent Cell ◽  
Gap Junction Channels ◽  
Amino Terminal ◽  
Type Iv ◽  
Green Fluorescent

Connexins oligomerize into hemichannels, traffic to the cell surface and dock with hemichannels from an adjacent cell to form intercellular gap junction channels. Pulsechase studies have revealed that connexins are subject to a short half-life ranging from 1- 5 hrs. To examine the mechanisms involved in connexin trafficking, gap junction assembly and internalization in living cells we fused green fluorescent protein (GFP) to the amino or carboxyl terminus of full length connexins (Cx). When cDNA encoding Cx43-GFP was transfected into communication-competent NRK cells, Cx43-negative MDCK cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into functional gap junctions (Figure 1). Most notably, the fusion of GFP to the amino terminal of Cx43 (GFP-Cx43) or Cx32 (GFP-Cx32) did not inhibit the co-translational insertion of these polytopic type IV connexins into the membrane and as a result gap junction plaques assembled at cellcell interfaces.

scholarly journals Conductance and Permeability of the Residual State of Connexin43 Gap Junction Channels

2002 ◽  
Vol 119 (2) ◽  
pp. 171-186 ◽  
Author(s):  
Feliksas F. Bukauskas ◽  
Angele Bukauskiene ◽  
Vytas K. Verselis
Keyword(s):  
Gap Junction ◽  
Fluorescent Protein ◽  
Single Channel ◽  
Electrical Coupling ◽  
Dye Transfer ◽  
Channel Pore ◽  
Gap Junction Channels ◽  
Gating Mechanism ◽  
Gated Channel ◽  
Green Fluorescent

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell–cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (Vj) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher Vjs that are negative on the side of gated hemichannel. The degree of rectification increases when Cl− is replaced by Asp− and decreases when K+ is replaced by TEA+. These data are consistent with an increased anionic selectivity of the residual state. The Vj-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast Vj-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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