全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
PLOS ONE  2012 

In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach

DOI: 10.1371/journal.pone.0033229

Full-Text   Cite this paper   Add to My Lib

Abstract:

Many essential cellular processes are affected by transmembrane H+ gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.

References

[1]  Tsien RY (1998) The Green Fluorescent Protein. Annu Rev Biochem 67: 509–544.
[2]  Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single lying cells with green fluorescent proteins. Proc Natl Acad Sci U S A 95: 6803–6808.
[3]  Miesenb?ck G, De Angelis GDA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature (London, U K) 6689: 192–195.
[4]  Bizzarri R, Arcangeli C, Arosio D, Ricci F, Faraci P, et al. (2006) Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies. Biophys J 90: 3300–3314.
[5]  Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45: 9639–9647.
[6]  Palková Z, Forstová J (2000) Yeast colonies synchronise their growth and development. J Cell Sci 113: 1923–1928.
[7]  Váchová L, Ku?erová H, Devaux F, Ulehlová M, Palková Z (2009) Metabolic diversification of cells during the development of yeast colonies. Environ Microbiol 11: 494–504.
[8]  Imai T, Ohno T (1995) Measurement of yeast intracellular pH by image processing and the change it undergoes during growth phase. J Biotechnol 38: 165–172.
[9]  Palková Z, Janderová B, Gabriel J, Zikánová B, Pospí?ek M, et al. (1997) Ammonia mediates communication between yeast colonies. Nature (London, U K) 6659: 532–535.
[10]  Sheff MA, Thorn KS (2004) Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21: 661–702.
[11]  Cormack BP, Bertram G, Egerton M, Gow NAR, Falkow S, et al. (1997) Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology 143: 303–311.
[12]  Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, et al. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22: 1567–1572.
[13]  Paiva S, Kruckeberg AL, Casal M (2002) Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae. Biochem J 363: 737–744.
[14]  Karagiannis J, Young PG (2001) Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe. J Cell Sci 114: 2929–2941.
[15]  Roermund CWT, de Jong M, IJlst L, van Marle J, Dansen TB, et al. (2004) The peroxisomal lumen in Saccharomyces cerevisiae is alkaline. J Cell Sci 117: 4231–4237.
[16]  Brett CL, Tukaye DN, Mukherjee S, Rao R (2005) The yeast endosomal Na+(K+)/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking. Mol Biol Cell 16: 1396–1405.
[17]  Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79: 2199–2208.
[18]  Olsen KN, Budde BB, Siegumfeldt H, Rechinger KB, Jakobsen M, et al. (2002) Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy. Appl Environ Microbiol 68: 4145–4147.
[19]  Disbrow GL, Hanover JA, Schlegel R (2005) Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH. J Virol 79: 5839–5846.
[20]  Mahon MJ (2011) pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Adv Biosci Biotechnol 2: 2156–8456.
[21]  Ashby MC, Ibaraki K, Henley JM (2004) It's green outside: tracking cell surface proteins with pH-sensitive GFP. Trends Neurosci 27: 257–261.
[22]  Granseth B, Odermatt B, Royle SJ, Lagnado L (2007) Clathrin-mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses. J Physiol 585: 681–686.
[23]  McDonald NA, Henstridge CM, Connolly CN, Irving AJ (2007) Generation and functional characterization of fluorescent, N-terminally tagged CB1 receptor chimeras for live-cell imaging. Mol Cell Neurosci 35: 237–248.
[24]  Nehrke K (2006) Intracellular pH measurements in vivo using green fluorescent protein variants. Methods Mol Biol (Totowa, NJ, U S) 351: 223–240.
[25]  Bagar T, Altenbach K, Read ND, Ben?ina M (2009) Live-cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe. Eukaryot Cell 8: 703–712.
[26]  Dechant R, Binda M, Lee SS, Pelet S, Winderickx J, et al. (2010) Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase. EMBO J 29: 2515–2526.
[27]  Orij R, Postmus J, Ter Beek A, Brul S, Smits GJ (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155: 268–278.
[28]  Braun NA, Morgan B, Dick TP, Schwappach B (2010) The yeast CLC protein counteracts vesicular acidification during iron starvation. J Cell Sci 123: 2342–2350.
[29]  Mare?ová L, Ho?ková B, Urbánková E, Chaloupka R, Sychrová H (2010) New applications of pHluorin - measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis. Yeast 6: 317–325.
[30]  Plant PJ, Manolson MF, Grinstein S, Demaurex N (1999) Alternative mechanisms of vacuolar acidification in H+-ATPase-deficient yeast. J Biol Chem 274: 37270–37279.
[31]  Bour-Dill C, Marie-Pierre G, Jean-Louis M, Marchal S, Guillemin F (2000) Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis. Cytometry 39: 16–25.
[32]  Varlamov O, Somwar R, Cornea A, Kievit P, Grove KL, et al. (2010) Single-cell analysis of insulin-regulated fatty acid uptake in adipocytes. Am J Physiol Endocrinol Metab 62: E486–E496.
[33]  Davies L, Satre M, Martin JB, Gross JD (1993) The target of ammonia action in dictyostelium. Cell 75: 321–327.
[34]  Moseyko N, Feldman LJ (2001) Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana. Plant Cell Environ 24: 557–563.
[35]  Lasorsa FM, Scarcia P, Erdmann R, Palmieri F, Rottensteiner H, et al. (2004) The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes. Biochem J 381: 581–585.
[36]  Matsuyama S, Llopis J, Deveraux QL, Tsien RY, Reed JC (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2: 318–325.
[37]  Mukherjee S, Kallay L, Brett CL, Rao R (2006) Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking. Biochem J 398: 97–105.
[38]  Jankowski A, Kim JH, Collins RF, Daneman R, Walton P, et al. (2001) In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHIuorin. J Biol Chem 276: 48748–48763.
[39]  Palková Z, Devaux F, ?i?icová M, Mináriková L, Le Crom S, et al. (2002) Ammonia Pulses and Metabolic Oscillations Guide Yeast Colony Development. Mol Biol Cell 13: 3901–3914.
[40]  Gori K, Mortensen HD, Arneborg N, Jespersen L (2007) Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species. J Dairy Sci 90: 5032–5041.
[41]  Bogonez E, Machado A Satrustegui J (1983) Ammonia accumulation in acetate-growing yeast. Biochim Biophys Acta 733: 234–241.
[42]  Palková Z, Váchová L, Gásková D, Kucerová H (2009) Synchronous plasma membrane electrochemical potential oscillations during yeast colony development and aging. Mol Membr Biol 26: 228–35.
[43]  Desai MJ, Armstrong DW (2003) Separation, identification, and characterization of microorganisms by capillary electrophoresis. Microbiol Mol Biol Rev 67: 38–51.
[44]  Xiong G, Aras O, Shet A, Key NS, Arriaga EA (2007) Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection. Analyst 128: 581–588.
[45]  ?i?icová M, Ku?erová H, Váchová L, Palková Z (2007) Association of putative ammonium exporters Ato with detergent-resistant compartments of plasma membrane during yeast colony development: pH affects Ato1p localisation in patches. Biochim Biophys Acta, Biomembr 1768: 1170–1178.
[46]  Gietz RD, Schiesti RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11: 355–360.
[47]  Wach A (1996) PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12: 259–265.
[48]  Preston RA, Murphy RF, Jones EW (1989) Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proc Natl Acad Sci U S A 86: 7027–7031.
[49]  Ali R, Brett CL, Mukherjee S, Rao R (2004) Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast. J Biol Chem 279: 4498–4506.
[50]  Wojdyr M (2010) Fityk: a general-purpose peak fitting program. J Appl Crystallogr 43: 1126–1128.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133