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

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.

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Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

Biochemical Journal ◽

10.1042/bj3490281 ◽

2000 ◽

Vol 349 (1) ◽

pp. 281-287 ◽

Cited By ~ 7

Author(s):

Patricia E. M. MARTIN ◽

James STEGGLES ◽

Claire WILSON ◽

Shoeb AHMAD ◽

W. Howard EVANS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gap Junctions ◽

Gap Junction ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Lucifer Yellow ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

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

Microscopy and Microanalysis ◽

10.1017/s1431927600018420 ◽

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.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells

Enzyme and Microbial Technology ◽

10.1016/j.enzmictec.2010.08.004 ◽

2011 ◽

Vol 48 (1) ◽

pp. 13-18 ◽

Cited By ~ 1

Author(s):

Wen-Hsin Lo ◽

Chi-Yuan Chen ◽

Chia-Ni Yeh ◽

Chin-Yu Lin ◽

Yu-Chen Hu

Keyword(s):

Green Fluorescent Protein ◽

Protein Expression ◽

Mammalian Cells ◽

Green Fluorescent Protein Expression ◽

Fluorescent Protein ◽

Green Fluorescent ◽

Expression In Mammalian Cells

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Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

The EMBO Journal ◽

10.1002/j.1460-2075.1996.tb00656.x ◽

1996 ◽

Vol 15 (12) ◽

pp. 2935-2943 ◽

Cited By ~ 48

Author(s):

F. Hanakam ◽

R. Albrecht ◽

C. Eckerskorn ◽

M. Matzner ◽

G. Gerisch

Keyword(s):

Plasma Membrane ◽

Green Fluorescent Protein ◽

Real Time ◽

Fluorescent Protein ◽

Ph Sensor ◽

Sensor Protein ◽

Green Fluorescent

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Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

The Journal of Cell Biology ◽

10.1083/jcb.139.6.1465 ◽

1997 ◽

Vol 139 (6) ◽

pp. 1465-1476 ◽

Cited By ~ 167

Author(s):

Norio Sakai ◽

Keiko Sasaki ◽

Natsu Ikegaki ◽

Yasuhito Shirai ◽

Yoshitaka Ono ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Green Fluorescent Protein ◽

Nmda Receptor ◽

Fusion Protein ◽

Fluorescent Protein ◽

Living Cells ◽

Kinase C ◽

Gfp Fusion Protein ◽

Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

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Subcellular redistribution of the renal betaine transporter during hypertonic stress

AJP Cell Physiology ◽

10.1152/ajpcell.00021.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1091-C1100 ◽

Cited By ~ 26

Author(s):

Stephen A. Kempson ◽

Vaibhave Parikh ◽

Lixuan Xi ◽

Shaoyou Chu ◽

Marshall H. Montrose

Keyword(s):

Plasma Membrane ◽

Posttranscriptional Regulation ◽

Fluorescent Protein ◽

Mdck Cells ◽

Live Cells ◽

Hypertonic Medium ◽

Hypertonic Stress ◽

Antibody Staining ◽

Renal Inner Medulla ◽

Green Fluorescent

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.

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Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein

Journal of Cell Science ◽

10.1242/jcs.112.16.2705 ◽

1999 ◽

Vol 112 (16) ◽

pp. 2705-2714

Author(s):

E.M. Burns ◽

L. Christopoulou ◽

P. Corish ◽

C. Tyler-Smith

Keyword(s):

Green Fluorescent Protein ◽

Mammalian Cells ◽

Quantitative Measurement ◽

Fluorescent Protein ◽

Cultured Cells ◽

Chromosome Loss ◽

Facs Analysis ◽

Fluorescent Cells ◽

Green Fluorescent ◽

Loss Rates

We have measured the mitotic loss rates of mammalian chromosomes in cultured cells. The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not. The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis. Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells. The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells.

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Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells—A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

Pharmaceutics ◽

10.3390/pharmaceutics12090805 ◽

2020 ◽

Vol 12 (9) ◽

pp. 805

Author(s):

Christopher Janich ◽

Daniel Ivanusic ◽

Julia Giselbrecht ◽

Elena Janich ◽

Shashank Reddy Pinnapireddy ◽

...

Keyword(s):

Drug Delivery ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Nucleic Acid ◽

Fluorescent Protein ◽

Cationic Lipid ◽

Nucleic Acid Delivery ◽

Molecular Clone ◽

Green Fluorescent ◽

Hiv 1

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

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