Real-time measurement of endosomal acidification by a novel genetically encoded biosensor

Serresi, Michela; Bizzarri, Ranieri; Cardarelli, Francesco; Beltram, Fabio
February 2009
Analytical & Bioanalytical Chemistry;Feb2009, Vol. 393 Issue 4, p1123
Academic Journal
Genetically encoded fluorescent proteins are optimal reporters when used to monitor cellular processes as they can be targeted to any subcellular region by fusion to a protein of interest. Here, we present the pH-sensitive fluorescent protein E1GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E1GFP is a ratiometric pH indicator by emission with a pK close to 6.0. We describe an application of this novel pH reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein which is endowed with cell-penetrating properties, we were able to monitor multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network in real time. This represents a framework for the application of E1GFP to the in situ detection of pH changes involved in dynamic biological phenomena. [Figure not available: see fulltext.]


Related Articles

  • Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study. Shevchuk, Andrew I.; Hobson, Phil; Lab, Max J.; Klenerman, David; Krauzewicz, Nina; Korchev, Yuri E. // Pflugers Archiv European Journal of Physiology;Apr2008, Vol. 456 Issue 1, p227 

    We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection...

  • Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit. Sato, Miyuki; Sato, Ken; Fonarev, Paul; Huang, Chih-Jen; Liou, Willisa; Grant, Barth D. // Nature Cell Biology;Jun2005, Vol. 7 Issue 6, p559 

    Here we identify a new regulator of endocytosis called RME-6. RME-6 is evolutionarily conserved among metazoans and contains Ras-GAP (GTPase-activating protein)-like and Vps9 domains. Consistent with the known catalytic function of Vps9 domains in Rab5 GDP/GTP exchange, we found that RME-6 binds...

  • Contribution of the Plasma Membrane and Central Vacuole in the Formation of Autolysosomes in Cultured Tobacco Cells. Yano, Kanako; Matsui, Sumiko; Tsuchiya, Tomohiro; Maeshima, Masayoshi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Moriyasu, Yuji // Plant & Cell Physiology;Jul2004, Vol. 45 Issue 7, p951 

    Autolysosomes accumulate in tobacco cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We characterized these plant autolysosomes using fluorescent dyes and green fluorescent protein (GFP). Observation using the endocytosis markers, FM4-64 and...

  • Internalization of the Receptor for Advanced Glycation End Products (RAGE) is Required to Mediate Intracellular Responses. Sevillano, Natalia; Girón, María D.; Salido, Mercedes; Vargas, Alberto M.; Vilches, José; Salto, Rafael // Journal of Biochemistry;Jan2009, Vol. 145 Issue 1, p21 

    To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE–EGFP protein is...

  • Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine. Beckman, M.; Kihlmark, M.; Iverfeldt, K.; Hallberg, E. // Apoptosis;May2004, Vol. 9 Issue 3, p363 

    The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate...

  • Trafficking of Green Fluorescent Protein-Tagged SNARE Proteins in HSY Cells1. Takuma, Taishin; Arakawa, Toshiya; Okayama, Miki; Mizoguchi, Itaru; Tanimura, Akihiko; Tajima, Yoshifumi // Journal of Biochemistry;2002, Vol. 132 Issue 5, p729 

    SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and...

  • Levels and localization of mechanosensitive channel proteins in Bacillus subtilis. Wahome, Paul G.; Cowan, Ann E.; Setlow, Barbara; Setlow, Peter // Archives of Microbiology;May2009, Vol. 191 Issue 5, p403 

    Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock ≥0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is...

  • Comparison of G-Protein Coupled Receptor Desensitization-Related β- Arrestin Redistribution Using Confocal and Non-Confocal Imaging. Haasen, Dorothea; Wolff, Michael; Valler, Martin J.; Heilker, Ralf // Combinatorial Chemistry & High Throughput Screening;Jan2006, Vol. 9 Issue 1, p37 

    High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we compared a confocal and a non-confocal cellular HCS system,...

  • Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott-Aldrich Syndrome. Uchiyama, Toru; Adriani, Marsilio; Jagadeesh, G Jayashree; Paine, Adam; Candotti, Fabio // Molecular Therapy;Jun2012, Vol. 20 Issue 6, p1270 

    The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics