Regulation of secretory granule pH in insulin-secreting cells

2002 ◽  
Vol 283 (2) ◽  
pp. C429-C437 ◽  
Author(s):  
Linda S. Tompkins ◽  
Kevin D. Nullmeyer ◽  
Sean M. Murphy ◽  
Craig S. Weber ◽  
Ronald M. Lynch
Keyword(s):  
Secretory Granule ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Single Cells ◽  
Human Growth ◽  
Egfp Gene ◽  
Peptide Release ◽  
Insulin Secreting Cells ◽  
Green Fluorescent ◽  
Insulinoma Cells

Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ∼4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H+-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.

scholarly journals Secretory-granule dynamics visualized in vivo with a phogrin–green fluorescent protein chimaera

1998 ◽  
Vol 333 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Aristea E. POULI ◽  
Evaggelia EMMANOUILIDOU ◽  
Chao ZHAO ◽  
Christina WASMEIER ◽  
John C. HUTTON ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Pc12 Cells ◽  
Secretory Granule ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Dense Core ◽  
Β Cells ◽  
Green Fluorescent ◽  
Insulinoma Cells

To image the behaviour in real time of single secretory granules in neuroendocrine cells we have expressed cDNA encoding a fusion construct between the dense-core secretory-granule-membrane glycoprotein, phogrin (phosphatase on the granule of insulinoma cells), and enhanced green fluorescent protein (EGFP). Expressed in INS-1 β-cells and pheochromocytoma PC12 cells, the chimaera was localized efficiently (up to 95%) to dense-core secretory granules (diameter 200–1000 nm), identified by co-immunolocalization with anti-(pro-)insulin antibodies in INS-1 cells and dopamine β-hydroxylase in PC12 cells. Using laser-scanning confocal microscopy and digital image analysis, we have used this chimaera to monitor the effects of secretagogues on the dynamics of secretory granules in single living cells. In unstimulated INS-1 β-cells, granule movement was confined to oscillatory movement (dithering) with period of oscillation 5–10 s and mean displacement < 1 µm. Both elevated glucose concentrations (30 mM), and depolarization of the plasma membrane with K+, provoked large (5–10 µm) saltatory excursions of granules across the cell, which were never observed in cells maintained at low glucose concentration. By contrast, long excursions of granules occurred in PC12 cells without stimulation, and occurred predominantly from the cell body towards the cell periphery and neurite extensions. Purinergic-receptor activation with ATP provoked granule movement towards the membrane of PC12 cells, resulting in the transfer of fluorescence to the plasma membrane consistent with fusion of the granule and diffusion of the chimaera in the plasma membrane. These results illustrate the potential use of phogrin–EGFP chimeras in the study of secretory-granule dynamics, the regulation of granule–cytoskeletal interactions and the trafficking of a granule-specific transmembrane protein during the cycle of exocytosis and endocytosis.

scholarly journals Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein

2003 ◽  
Vol 77 (22) ◽  
pp. 12203-12210 ◽  
Author(s):  
Albert A. Rizvanov ◽  
Albert G. M. van Geelen ◽  
Sergey Morzunov ◽  
Elmer W. Otteson ◽  
Charlotte Bohlman ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Deer Mice ◽  
Anamnestic Response ◽  
Egfp Gene ◽  
Virus Challenge ◽  
Virus Particles ◽  
Green Fluorescent ◽  
Human Cmv ◽  
A Site

ABSTRACT A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.

Oligonucleotide Preparation Approach for Assembly of DNA Synthons

2019 ◽  
Vol 24 (6) ◽  
pp. 556-568
Author(s):  
Aleksei V. Yantsevich ◽  
Veronika V. Shchur ◽  
Sergey A. Usanov
Keyword(s):  
Fluorescent Protein ◽  
Solid Phase ◽  
Base Pairs ◽  
Egfp Gene ◽  
Standard Facility ◽  
Synthetic Dna ◽  
Pcr Product ◽  
Green Fluorescent ◽  
Inside Out ◽  
Spe Purification

An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40–60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Abstract Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Nicole Faust ◽  
Florencio Varas ◽  
Louise M. Kelly ◽  
Susanne Heck ◽  
Thomas Graf
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Neutrophil Granulocytes ◽  
Egfp Gene ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Myelomonocytic Cells ◽  
Green Fluorescent

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645-649 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B. M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Reporter Protein ◽  
Regulated Secretory Pathway ◽  
Green Fluorescent

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

scholarly journals Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

2015 ◽  
Vol 112 (39) ◽  
pp. 12181-12186 ◽  
Author(s):  
Chaoxian Bai ◽  
Yang Zhang ◽  
Xuejin Zhao ◽  
Yiling Hu ◽  
Sihai Xiang ◽  
...  
Keyword(s):  
Quantitative Method ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Gene Clusters ◽  
Regulatory Elements ◽  
Streptomyces Avermitilis ◽  
Systems Biotechnology ◽  
Synthetic Promoters ◽  
Expression Of Genes ◽  
Green Fluorescent

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

scholarly journals Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

1998 ◽  
Vol 9 (9) ◽  
pp. 2463-2476 ◽  
Author(s):  
Janis E. Lochner ◽  
Mary Kingma ◽  
Samuel Kuhn ◽  
C. Daniel Meliza ◽  
Bryan Cutler ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Plasminogen Activator ◽  
Axonal Transport ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Time Lapse ◽  
Growth Cones ◽  
Differentiated Cells ◽  
Tissue Plasminogen ◽  
Green Fluorescent

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

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