Chapter 12 - Fluorescence methods for analysis of interactions between Ca2+ signaling, lysosomes, and endoplasmic reticulum

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Abstract

The endoplasmic reticulum (ER) is both the major source of intracellular Ca2+ for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca2+ signaling by both releasing and sequestering Ca2+. Interactions between ER and other Ca2+-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca2+ signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca2+ signals, the ER, and lysosomes to be examined.

Section snippets

ER, Lysosomes, and Ca2+ Signaling

Experimental analyses of Ca2+ signaling have seen different membranes move in and out of the limelight. For many years, beginning with the first evidence that Ca2+ regulates cellular activities (Ringer, 1883), Ca2+ influx across the plasma membrane was assumed to be entirely responsible for increases in cytosolic free Ca2+ concentration ([Ca2+]c). These Ca2+ entry pathways are important, indeed the store-operated Ca2+ entry (SOCE) pathway is almost ubiquitous (Putney, 1997), but Ca2+ channels

Pharmacological Tools

Two widely used membrane-permeant inhibitors allow selective inhibition of SERCA. The plant sesquiterpene lactone, thapsigargin, irreversibly inhibits SERCA (Michelangeli and East, 2011, Sagara and Inesi, 1991), while cyclopiazonic acid causes reversible inhibition (Demaurex, Lew, & Krause, 1992). Each depletes the ER of Ca2+ as basal leaks proceed unopposed by Ca2+ pumping. Selective activation of IP3R is usually achieved by stimulation of receptors coupled to phospholipase C (Lopez Sanjurjo

Fluorescence Methods

Absorption of a photon by a fluorescent molecule moves an electron from its ground state (S0) to an excited singlet state (S2). Over a few ns, some of the absorbed energy is then dissipated before the electron returns (from S1) to its ground state, emitting a photon with less energy (longer wavelength) than the one that caused excitation (Lakowicz, 2006). It would be hard to over-state the impact of fluorescence methods in biology, and the reasons are numerous (Giepmans et al., 2006, Zhang

Fluorescence Tools for Analysis of Lysosomes

Our focus on Ca2+ signaling, ER, and lysosomes identifies the need for fluorescent probes for reporting [Ca2+] and organelle identity. Conventional, BAPTA-based Ca2+ indicators (e.g., fura 2, fluo 4, etc.) in their acetoxymethyl (AM) ester forms allow facile loading of cells with fluorescent reporters of [Ca2+]c. It is, however, necessary to optimize loading protocols to avoid compartmentalization of the indicator within organelles or its extrusion across the plasma membrane (Bootman, Rietdorf,

Ca2+ Signaling and Lysosomes: Tools and Practical Problems

The lumen of the lysosome is an exceptionally hostile environment in which to measure free [Ca2+]. The acidic pH (∼pH 4.5) (Ishida, Nayak, Mindell, & Grabe, 2013) massively reduces the Ca2+ affinity of indicators, and even small changes in pH, such as are expected to accompany Ca2+ uptake and/or release by lysosomes (Lopez Sanjurjo et al., 2013, Morgan and Galione, 2007), may substantially change the apparent affinity of the indicator. It thus becomes difficult to disentangle changes in pH from

Materials

  • 1.

    Calcium Calibration Buffer Kit #1 and fura 2-AM (Life Technologies, Paisley, UK). Pluronic F127 (Sigma, Poole, UK). Ionomycin (Merck Eurolab, Nottingham, UK)

  • 2.

    22-mm diameter round glass coverslips coated with poly-l-lysine

  • 3.

    HBS (HEPES-buffered saline): NaCl 135 mM, KCl 5.9 mM, MgCl2 1.2 mM, CaCl2 1.5 mM, HEPES 11.6 mM and glucose 11.5 mM, pH 7.3. Ca2+ is omitted from nominally Ca2+-free HBS, and replaced by BAPTA (10 mM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, Molekula, Dorset, UK) in Ca

High-throughput Analyses of Cytosolic Ca2+ Signals

Single-cell analyses unmask cellular heterogeneity and afford opportunities to examine subcellular Ca2+ signals, but they are not easily adapted to quantitative analyses of concentration-effect relationships or high-throughput screening. Rapid measurements of [Ca2+]c from cells grown in 96-well plates better meet these requirements (Tovey, Sun, & Taylor, 2006). Here we describe the use a FlexStation III 96-well fluorescence plate-reader equipped to allow up to three automated online additions

Tracking Interactions between Lysosomes and ER by Fluorescence Microscopy

Both lysosomes and ER are dynamic organelles (Figure 2(B)), and while electron microscopy provides an informative snapshot of their association in fixed cells (Kilpatrick et al., 2013), it cannot resolve dynamic interactions, and fixation may distort associations. But non-invasive tracking of lysosomes in live cells is challenging: fluorophore bleaching can limit opportunities to capture images for sufficient time and with sufficient temporal resolution; and automated tracking of small dynamic

Conclusions

A recurrent theme in Ca2+ signaling is the importance of spatially organized Ca2+ signals (Berridge, Bootman, & Roderick, 2003). It is becoming increasingly recognized that interactions between intracellular membranes, often facilitated by scaffold proteins or tethers, play important roles in both shaping and decoding these Ca2+ signals (Lam and Galione, 2013, Prinz, 2014). Lysosomes are relative latecomers to the community of Ca2+ signaling organelles (Morgan et al., 2011), but there is

Acknowledgments

Supported by the Biotechnology and Biological Sciences Research Council (L0000075). CWT is a Wellcome Trust Senior Investigator (101844). CIL-S was supported by studentships from Caixa Galicia Foundation and Obra Social La Caixa, Spain.

References (81)

  • P. Huang et al.

    P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH

    Journal of Biological Chemistry

    (2014)
  • V. Konieczny et al.

    Spatial organization of intracellular Ca2+ signals

    Seminars in Cell & Developmental Biology

    (2012)
  • A. Lam et al.

    The endoplasmic reticulum and junctional membrane communication during calcium signaling

    Biochimica et Biophysica Acta

    (2013)
  • H.C. Lee

    Nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated calcium signaling

    Journal of Biological Chemistry

    (2005)
  • R.M. Lemons et al.

    Mediated calcium transport by isolated human fibroblast lysosomes

    Journal of Biological Chemistry

    (1991)
  • W. Li et al.

    Membrane-permeant esters of inositol polyphosphates, chemical syntheses and biological applications

    Tetrahedron

    (1997)
  • J. Liu et al.

    Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators

    Bioorganic and Medicinal Chemistry Letters

    (2001)
  • J. Lytton et al.

    Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps

    Journal of Biological Chemistry

    (1992)
  • J.J. Mackrill

    Ryanodine receptor calcium channels and their partners as drug targets

    Biochemical Pharmacology

    (2010)
  • V. Marshansky et al.

    The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function

    Current Opinion in Cell Biology

    (2008)
  • E. Meijering et al.

    Methods for cell and particle tracking

    Methods in Enzymology

    (2012)
  • S. Patel et al.

    Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

    Trends in Cell Biology

    (2010)
  • S. Patel et al.

    The endo-lysosomal system as an NAADP-sensitive acidic Ca2+ store: role for the two-pore channels

    Cell Calcium

    (2011)
  • Y. Sagara et al.

    Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations

    Journal of Biological Chemistry

    (1991)
  • J. Shuai et al.

    Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

    Cell Calcium

    (2005)
  • C.W. Taylor et al.

    Intracellular Ca2+ channels – a growing community

    Molecular and Cellular Endocrinology

    (2012)
  • X. Wang et al.

    TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes

    Cell

    (2012)
  • T. Yoshimori et al.

    Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells

    Journal of Biological Chemistry

    (1991)
  • M.J. Berridge

    Elementary and global aspects of calcium signalling

    Journal of Physiology

    (1997)
  • M.J. Berridge et al.

    Calcium signalling: dynamics, homeostasis and remodelling

    Nature Reviews: Molecular Cell Biology

    (2003)
  • M.J. Berridge et al.

    The versatility and universality of calcium signalling

    Nature Reviews: Molecular Cell Biology

    (2000)
  • E. Betzig et al.

    Imaging intracellular fluorescent proteins at nanometer resolution

    Science

    (2006)
  • M.D. Bootman et al.

    Ca2+-sensitive fluorescent dyes and intracellular Ca2+ imaging

  • P.J. Calcraft et al.

    NAADP mobilizes calcium from acidic organelles through two-pore channels

    Nature

    (2009)
  • K.A. Christensen et al.

    pH-dependent regulation of lysosomal calcium in macrophages

    Journal of Cell Science

    (2002)
  • X.P. Dong et al.

    TRP channels of intracellular membranes

    Journal of Neurochemistry

    (2010)
  • S. Drose et al.

    Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases

    Journal of Experimental Biology

    (1997)
  • J.H. Exton

    Mechanisms involved in α-adrenergic phenomena: role of calcium ions in actions of catecholamines in liver and other tissues

    American Journal of Physiology

    (1980)
  • J.R. Friedman et al.

    Endoplasmic reticulum-endosome contact increases as endosomes traffic and mature

    Molecular Biology of the Cell

    (2013)
  • B.N. Giepmans et al.

    The fluorescent toolbox for assessing protein location and function

    Science

    (2006)

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