Hücre İçi Serbest Ca2+ Konsantrasyon Dinamiğinin Floresans Yöntemler ile İncelenmesi

Yıl 2016, Cilt: 25 Sayı: 3, 319 - 334, 30.09.2016

Figen Çiçek , İsmail Günay

https://doi.org/10.17827/aktd.237675

Öz

Hücre membranına gelen uyarıların bir çoğu, sitoplazmik serbest Ca+2 derişiminde ([Ca+2]S) artışa neden olur. Ca+2'un sitozoldeki konsantrasyonunun oldukça düşük (≈10-7 M), hücre dışı sıvıdaki ve endoplazmik retikulumdaki (ER) (≈10-3 M) konsantrasyonunun ise yüksek olmasından dolayı, pek çok hücrede Ca+2 hücre içi sinyal molekülü ve ikinci haberci olarak kullanılır. Ca+2’u, plazma ve ER membranlarından sitozole doğru iten böyle büyük bir elektro-kimyasal gradiyent nedeni ile plazma ya da ER membranlarında bulunan Ca+2 kanallarının geçici olarak açılmasını sağlayacak bir sinyal, Ca+2 un hızla sitozole akmasına ve [Ca+2]S’nun 10-20 kat artmasına neden olur. Böylece hücre içinde bulunan Ca+2’a duyarlı proteinler aktive olarak bir çok hücresel fonksiyonun yerine getirilmesi sağlanır. Örneğin gen ekspresyonu, hücre çoğalması, bölünmesi, apoptoz ve ayrıca kas hücrelerinde kasılma, salgı hücrelerinde degranülasyon ve sinirsel iletim gibi olayların tetiklenmesinde Ca+2 kilit rol oynar. Bu nedenle [Ca+2]S’nun dinamik olarak ölçülebilmesi, hücrede gerçekleşen pek çok sinyalizasyon mekanizmasının anlaşılmasında önemli bir yer tutar. Floresans görüntüleme teknikleri, Ca+2’un hücre içinde uzay/zaman değişim desenlerini takip edilebilir duruma getirmiştir ve bu sayede bu tekniklerin kullanıldığı yöntemler de son yıllarda gelişerek artmıştır. Özellikle floresans boyaların kullanım kolaylıkları ve hücre hemostazına müdahalenin diğer metotlara göre minimum oranda kalması, bu metotların kullanımının önünü açmıştır.  

Anahtar Kelimeler

Floresans, Ca+2 görüntüleme, IP3 sinyal yolağı

Investigation of Intracellular Free Ca2+ Concentration Dynamics with Fluorescence Methods

Öz

Most of the extracellular stimulus arrive to the cell membrane result with the increase in cytoplasmic free Ca+2 concentration [Ca+2]i. Because of the huge Ca+2 concentration differences between the cytoplasm (≈10-7 M) and extracellular fluid and endoplasmic reticulum (ER - which is the major Ca+2 storage organelle in especially non electrically excitable cells) (≈10-3 M), a large electro-chemical gradient repel Ca+2 to the plasma or ER. Therefore a signal which temporarily opens Ca+2 channels, induce a fast influx of Ca+2 through the cytosol and increase its concentration about 10-20 fold. At this organization free Ca+2 functions as an intracellular signalling molecule and a second messenger. In this way many intracellular signalling proteins activated and cellular functions like gene expression, cell proliferation and division, apoptosis, and also myocyte contraction, endocrine cell degranulation and neuronal transmission are regulated. Thus, the key role of Ca+2 in many intracellular process, makes the dynamic measurements necessary for an understanding of the signalling mechanisms. Fluorescence imaging techniques make possible of monitoring the spatiotemporal Ca+2 response patterns in cytoplasm. In the last decades, especially their ease of loading, and minimum manipulations to the cell homeostasis, make these techniques unique with respect to the other methods.

Anahtar Kelimeler

Fluorescence, Ca+2 imaging, IP3 signaling pathway

Kaynakça

• Bootman MD, Collins TJ, Peppiatt CM, Prothero LS, MacKenzie L, De Smet P et al. Calcium signalling--an overview. Semin Cell Dev Biol. 2001;12:3-10.
• Berridge MJ. Neuronal calcium signaling. Neuron. 1998;21:13-26.
• Verkhratsky A. Calcium and cell death. Subcell Biochem. 2007;45:465-80.
• Verkhratsky A, Petersen OH. The endoplasmic reticulum as an integrating signalling organelle: from neuronal signalling to neuronal death. Eur J Pharmacol. 2002;447:141-54.
• Falcke M. Reading the patterns in living cells - the physics of Ca2+ signaling. Advances in Physics. 2004;53:255-440.
• Berridge MJ. Inositol trisphosphate and calcium signalling mechanisms. Biochim Biophys Acta. 2009;1793:933-940.
• Cicek FA, Ozgur EO, Ozgur E, Ugur M. The interplay between plasma membrane and endoplasmic reticulum Ca(2+)ATPases in agonist-induced temporal Ca(2+) dynamics. J Bioenerg Biomembr. 2014;46:503-10.
• Guerrero-Hernandez A, Dagnino-Acosta A, Verkhratsky A. An intelligent sarco-endoplasmic reticulum Ca2+ store: release and leak channels have differential access to a concealed Ca2+ pool. Cell Calcium. 2010;48:143-9.
• Brini M, Carafoli E. Calcium pumps in health and disease. Physiol Rev. 2009;89:1341-78.
• Eisner V, Csordas G, Hajnoczky G. Interactions between sarco-endoplasmic reticulum and mitochondria in cardiac and skeletal muscle: pivotal roles in Ca(2)(+) and reactive oxygen species signaling. J Cell Sci. 2013;126:2965-78.
• Bossuyt J, Bers DM. Visualizing CaMKII and CaM activity: a paradigm of compartmentalized signaling. J Mol Med (Berl). 2013;91:907-16.
• Swillens S, Champeil P, Combettes L, Dupont G. Stochastic simulation of a single inositol 1,4,5-trisphosphate-sensitive Ca2+ channel reveals repetitive openings during 'blip-like' Ca2+ transients. Cell Calcium. 1998;23:291-302.
• Dickinson GD, Parker I. Temperature dependence of IP3-mediated local and global Ca2+ signals. Biophys J. 2013;104:386-95.
• Stern MD, Rios E, Maltsev VA. Life and death of a cardiac calcium spark. J Gen Physiol. 2013;142:257-74.
• Cheng H, Lederer WJ. Calcium sparks. Physiol Rev. 2008;88:1491-1545.
• Hove-Madsen L, Llach A, Bayes-Genis A, Roura S, Rodriguez Font E, Aris A et al. Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes. Circulation. 2004;110:1358-63.
• Eisner DA, Trafford AW. A sideways look at sparks, quarks, puffs and blips. J Physiol. 1996;497(Pt 1):2.
• Endo M. Calcium-induced calcium release in skeletal muscle. Physiol Rev. 2009;89:1153-76.
• Dupont G. Modeling the intracellular organization of calcium signaling. Wiley Interdiscip Rev Syst Biol Med. 2014;6:227-37.
• Lakowicz JR. Principles of Fluorescence Spectroscopy, 3rd ed., New York, Springer, 2006.
• Borle AB. An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells. Environ Health Perspect. 1990;84:45-56.
• Blinks JR, Wier WG, Hess P, Prendergast FG. Measurement of Ca2+ concentrations in living cells. Prog Biophys Mol Biol. 1982;40:1-114.
• Beeler T. Oxidation of sulfhydryl groups and inhibition of the (Ca2+ + Mg2+)-ATPase by arsenazo III. Biochim Biophys Acta. 1990;1027:264-67.
• Alvarez J, Montero M. Measuring [Ca2+] in the endoplasmic reticulum with aequorin. Cell Calcium. 2002;32:251-60.
• Solovyova N, Verkhratsky A. Monitoring of free calcium in the neuronal endoplasmic reticulum: an overview of modern approaches. J Neurosci Methods. 2002;122:1-12.
• Shimomura O. Luminescence of aequorin is triggered by the binding of two calcium ions. Biochem Biophys Res Commun. 1995;211:359-63.
• Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J. 1995;14:5467-75.
• Brini M, Pasti L, Bastianutto C, Murgia M, Pozzan T, Rizzuto R. Targeting of aequorin for calcium monitoring in intracellular compartments. J Biolumin Chemilumin. 1994;9:177-184.
• Kendall JM, Badminton MN, Sala-Newby GB, Wilkinson GW, Campbell AK. Agonist-stimulated free calcium in subcellular compartments: delivery of recombinant aequorin to organelles using a replication deficient adenovirus vector. Cell Calcium. 1996;19:133-42.
• Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44.
• Dickson RM, Cubitt AB, Tsien RY, Moerner WE. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature. 1997;388:355-8.
• Loew LM. Where does all the PIP2 come from? J Physiol. 2007;582:945-51.
• Hirose K, Kadowaki S, Tanabe M, Takeshima H, Lino M. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science. 1999;284:1527-30.
• Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997;388:882-7.
• Takanishi CL, Bykova EA, Cheng W, Zheng J. GFP-based FRET analysis in live cells. Brain Res. 2006;1091:132-9.
• Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-50.
• Paredes RM, Etzler JC, Watts LT, Zheng W, Lechleiter JD. Chemical calcium indicators. Methods. 2008;46:143-51.
• Takahashi A, Camacho P, Lechleiter JD, Herman B. Measurement of intracellular calcium. Physiol Rev. 1999;79:1089-1125.
• Mason WT. Fluorescent and luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantative Real-Time Analysis. 2nd ed.. San Diego ; London, Academic Press, 1999.
• Greimers R, Trebak M, Moutschen M, Jacobs N, Boniver J. Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets. Cytometry. 1996;23:205-17.
• Pande G, Kumar NA, Manogaran PS. Flow cytometric study of changes in the intracellular free calcium during the cell cycle. Cytometry. 1996;24:55-63.
• Tuncay E, Okatan EN, Vassort G, Turan B. ss-blocker timolol prevents arrhythmogenic Ca(2)(+) release and normalizes Ca(2)(+) and Zn(2)(+) dyshomeostasis in hyperglycemic rat heart. PLoS One. 2013;8:e71014.
• Kao JP, Harootunian AT, Tsien RY. Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem. 1989;264:8179-84.
• Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium. 2000;27:97-106.
• Brandes R, and Bers DM. Simultaneous measurements of mitochondrial NADH and Ca(2+) during increased work in intact rat heart trabeculae. Biophys J. 2002;83:587-604.
• Hayashi H, and Miyata H. Fluorescence imaging of intracellular Ca2+. J Pharmacol Toxicol Methods. 1994;31:1-10.
• Tsien RY. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature. 1981;290:527-8.
• Jobsis PD, Rothstein EC, and Balaban RS. Limited utility of acetoxymethyl (AM)-based intracellular delivery systems, in vivo: interference by extracellular esterases. J Microsc. 2007;226:74-81.
• Williams DA. Mechanisms of calcium release and propagation in cardiac cells. Do studies with confocal microscopy add to our understanding? Cell Calcium. 1993;14:724-35.
• Miyazaki S. Inositol trisphosphate receptor mediated spatiotemporal calcium signalling. Curr Opin Cell Biol. 1995;7:190-6.