Imaging approaches to measuring lysosomal calcium.

CHAPTER

Imaging approaches to measuring lysosomal calcium

9

Anthony J. Morgan1, Lianne C. Davis, Antony Galione Department of Pharmacology, University of Oxford, Oxford, UK 1

Corresponding author: E-mail: [email protected]

CHAPTER OUTLINE 1. Endolysosomal Ca2D .......................................................................................... 160 1.1 Endolysosomal Ca2þ: Roles and Regulation ........................................... 160 1.1.1 Role of Ca2þ ..................................................................................... 160 1.2 Endolysosomal Ca2þ Homeostasis......................................................... 161 2. Assessing Organelle Ca2D: General Strategies..................................................... 161 2.1 Global Cytosolic Ca2þ Measurements .................................................... 162 2.2 Perivesicular Ca2þ Measurements......................................................... 163 2.3 Luminal Ca2þ Measurements ............................................................... 165 2.3.1 Total Ca2þ ........................................................................................ 165 2.3.2 Free Ca2þ ........................................................................................ 165 3. Assessing Endolysosomal Ca2D: Specific Strategies ............................................ 168 3.1 Indirect Monitoring with Cytosolic Ca2þ Indicators ................................. 169 3.1.1 Agents that target acidic Ca2þ stores................................................. 170 3.1.2 Ca2þ-indicator loading ...................................................................... 172 3.1.3 Ca2þ measurements ......................................................................... 173 3.1.4 Indirect measurements: pitfalls ......................................................... 175 3.1.5 Conclusion ....................................................................................... 176 3.2 Direct Luminal Recording .................................................................... 176 3.2.1 Luminal pH ...................................................................................... 176 3.2.2 Is pHL always a problem?.................................................................. 177 3.3 Luminal Recording: Practicalities ......................................................... 180 3.3.1 Targeting indicators to acidic vesicles................................................ 180 3.3.2 Resting or dynamic [Ca2þ] changes?................................................. 182 3.3.3 Calibration and correcting for pHL ..................................................... 182 3.3.4 Luminal Ca2þ protocol ...................................................................... 184 3.3.5 Conclusions...................................................................................... 189

Methods in Cell Biology, Volume 126, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2014.10.031 © 2015 Elsevier Inc. All rights reserved.

159

160

CHAPTER 9 Imaging approaches to measuring lysosomal calcium

4. Final Remarks .................................................................................................... 189 References ............................................................................................................. 189

Abstract Endolysosomes are emerging as key players that generate as well as respond to intracellular Ca2þ signals. The role of Ca2þ in modulating acidic organelle function has long been recognized, but it is now emerging that acidic organelles also act as intracellular Ca2þ stores; they actively sequester Ca2þ in their lumina and release it to the cytosol upon activation of endolysosomal Ca2þ channels. This local Ca2þ signal is crucial for endolysosomal function and/or global Ca2þ signaling. Importantly, defects in endolysosomal Ca2þ are associated with disease. This chapter discusses several complimentary approaches to monitor endolysosomal Ca2þ, with particular emphasis on the inherent pitfalls that can plague the unwary.

1. ENDOLYSOSOMAL Ca2D 1.1 ENDOLYSOSOMAL Ca2D: ROLES AND REGULATION Before focusing on detailed methodologies, it is valuable to first briefly consider: (1) Why is Ca2þ important for endolysosomal biology? and (2) How is endolysosomal Ca2þ regulated?

1.1.1 Role of Ca2þ What distinguishes endolysosomal vesicles from most other organelles is, of course, their characteristic Hþ content, but other luminal ions (e.g., Cl, Ca2þ) have emerged more recently as crucial contributors to vesicle physiology (Morgan, Platt, LloydEvans, & Galione, 2011; Stauber & Jentsch, 2013). Indeed, dysfunction in endolysosomal Cl or Ca2þ homeostasis underlies (or exacerbates) various mammalian diseases (Coen et al., 2012; Morgan et al., 2011; Stauber & Jentsch, 2013) and so this is no backwater of cell biology. As this entire volume attests, the endolysosomal system is more than the cellular “stomach” but a pleiotropic “hub” that coordinates multiple biochemical pathways. Such coordination requires communication with other parts of the cell, and endolysosomes can send and receive signals via the messenger, Ca2þ. Acidic vesicles play a role in diverse cellular pathways and Ca2þ impinges upon many of these different aspects. First, acidic vesicles can act as targets for Ca2þ: e.g., Ca2þ stimulates exocytosis (secretory vesicles); Ca2þ is essential for trafficking and fusion (endolysosomes) (Ruas et al., 2010) or autophagy (autophagic vacuoles) (Lu et al., 2013). Alternatively, acidic vesicles act as sources of Ca2þ to regulate unique downstream processes: e.g., endolysosomal Ca2þ regulates plasma membrane excitability (Brailoiu, Brailoiu, et al., 2009; Calcraft et al., 2009), cell differentiation (Brailoiu, et al., 2006; Aley et al., 2010; Zhang, Lu, & Yue, 2013), and triggers global Ca2þ signals by recruiting the ER (endoplasmic reticulum) Ca2þ store (Morgan et al., 2011). There are even instances when acidic vesicles are both source and target: in some cell types, the Ca2þ for exocytosis can be provided by the secretory

2. Assessing Organelle Ca2þ: General Strategies

vesicle itself in an example of “paracrine” organelle signaling (Davis et al., 2008; Davis et al., 2012; Mitchell, Lai, & Rutter, 2003; Santodomingo et al., 2008).

1.2 ENDOLYSOSOMAL Ca2D HOMEOSTASIS What are the details of Ca2þ homeostasis in the endolysosomal system? The general principles of intracellular Ca2þ signaling have been thoroughly reviewed elsewhere (Berridge, Bootman, & Roderick, 2003; Berridge, 2009), but in essence the low cytosolic [Ca2þ] (w100 nM) is maintained by the concerted action of Ca2þ pumps and exchangers in both the plasma membrane and intracellular Ca2þ stores (including the ER and the Golgi). A Ca2þ store is operationally defined as any organelle that accumulates Ca2þ in its lumen (usually to millimolar levels) but then releases this Ca2þ to the cytosol upon demand by the opening of Ca2þ channels. In essentials, the endolysosomal system operates on the same principles as the archetypal ER Ca2þ store, even though the details differ. In the simplest model, Ca2þ-storing organelles require some Ca2þ-uptake mechanism(s), luminal Ca2þ-binding sites, and Ca2þ-release mechanism(s). In the ER, this could be represented by SERCA (sarco-endoplasmic reticulum Ca2þ-ATPase), calsequestrin, and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). However, the equivalent molecular details of the endolysosomes are less clearly defined. The precise mechanism of Ca2þ uptake into endolysosomes in mammalian cells is currently unknown. Similar to the better understood plant and yeast acidic vesicles (i.e., vacuoles) (Pittman, 2011), mammalian Ca2þ-uptake depends upon the energy of the Hþ-gradient and has, therefore, been surmised as some form of Ca2þ/Hþ exchange (CHX), rather than a SERCA-mediated process (Figure 1) (see (Morgan et al., 2011) for detailed discussion). Luminal Ca2þ must be predominantly stored in a complexed, bound form (to avoid precipitation at high concentrations of free Ca2þ), but again, we are unclear as to the Ca2þ-binding matrix (it could be a polyanion e.g., polyphosphate, or Ca2þ-binding proteins). By contrast, we are making more in-roads into identifying potential Ca2þ-release channels. Just as the ER/SR (endo/ sarcoplasmic reticulum) possesses multiple channel families stimulated by second messengers (IP3 or cyclic ADP-ribose), so too is endolysosomal Ca2þ release stimulated by ligands (Figure 1) such as nicotinic acid adenine dinucleotide phosphate (NAADP) or PI(3,5)P2 (Phosphatidylinositol 3,5-bisphosphate), acting via two-pore channels (TPCs) (Brailoiu, Churamani, et al., 2009; Calcraft et al., 2009; Jha, Ahuja, Patel, Brailoiu, & Muallem, 2014; Wang et al., 2012; Zong et al., 2009) or mucolipins (Dong et al., 2010; Lelouvier & Puertollano, 2011). Although there is still some controversy surrounding the role of TPCs (discussed (Morgan & Galione, 2014)), the majority of the literature implicates TPCs in endolysosomal Ca2þ homeostasis. The role of NAADP and TPCs in endolysosomal pathophysiology is an ever-growing field.

2. ASSESSING ORGANELLE Ca2D: GENERAL STRATEGIES There are multiple reasons why one needs to monitor the Ca2þ levels within a particular organelle: (1) How does a particular organelle contribute to the time-course of

161

162


FIGURE 1 Ion Homeostasis in Acidic Organelles. A simplistic scheme of aspects of Hþ and Ca2þ homeostasis in an acidic vesicle. The V-HþATPase pumps Hþ into and acidifies the organelle lumen. Ca2þ is transported into the lumen by exploiting the energy of the Hþ gradient in some version of Ca2þ/Hþ exchange (the transporter is not known in mammalian cells, and might even represent the net result of two consecutive reactions: Naþ/Hþ then Naþ/Ca2þ exchange (Morgan et al. 2011)). Ca2þ is released through Ca2þ-permeable channels such as two-pore channels (TPCs) gated by NAADP (or TRPML1 gated by PI(3,5)P2), although this is controversial (Morgan & Galione, 2014). (See color plate)

complex Ca2þ signals? (2) What is the Ca2þ concentration within an organelle under certain experimental or pathological conditions? (3) How does luminal Ca2þ act as a regulator of endolysosomal function? These are obviously interrelated and not mutually exclusive scenarios.

2.1 GLOBAL CYTOSOLIC Ca2D MEASUREMENTS Can we obtain any information from simple cytosolic Ca2þ signals? Although measuring cytosolic Ca2þ signals is technically straightforward these days with optical reporters such as fura-2 (Takahashi, Camacho, Lechleiter, & Herman, 1999), interpreting them is anything but easy. Multiple Ca2þ sources and multiple channels can simultaneously contribute to the cytosolic Ca2þ signal, such that disentangling each input is far from trivial. Nonetheless, some information can be gained. An artificial, but useful, strategy is to rapidly discharge the Ca2þ content of a given Ca2þ store and monitor how much Ca2þ appears in the cytosol (Figure 2; see below). Simplistically, the more Ca2þ inside the store, the bigger and more rapid the cytosolic Ca2þ spike. Alternatively, we can monitor stimulus-induced cytosolic signals and ask what is the contribution of a given store? Therefore, cytosolic Ca2þ is monitored in the presence of pharmacological (or genetic) blockade of a given Ca2þ source (e.g., Ca2þ-store depletion, Ca2þ-channel blocker, siRNA). Both can be an effective approaches, but should be designed and interpreted cautiously: for example, depleting a Ca2þ source is a major perturbation and may have unexpected knock-on effects. A related complication is that the different Ca2þ stores often communicate with one another (Figure 3; see below) and targeting


FIGURE 2 Approaches for assessing endolysosomal Ca2D content. Two major strategies for assessing the Ca2þ content. The Indirect method uses a Ca2þ dye in the cytosolic compartment (this is usually free in the cytosol but could also be tethered to the extralysosomal membrane on its outer face). Ca2þ is released from the organelle lumen and the size of the resultant Ca2þ spike is qualitatively proportional to the content (with caveatsdsee text). The Direct method relies on a Ca2þ indicator loaded into the endolysosomal lumen to give a quantitative value of the luminal Ca2þ concentration. (See color plate)

one organelle may unavoidably affect a neighboring organelle, resulting in misinterpretation. We shall return to these issues later in the context of determining organelle Ca2þ content.

2.2 PERIVESICULAR Ca2D MEASUREMENTS A better approach is to monitor cytosolic [Ca2þ] not globally, but locally, immediately around the vesicle itself. This has the advantage of (1) avoiding the problems of luminal recording (see below); (2) potentially recording small or local Ca2þ signals that are not otherwise detectable in global cytosolic signals (a propos when one considers the small size of the endolysosomal Ca2þ pool compared to the vast Ca2þ reservoirs that are the ER or extracellular milieu). Though discrete, these local Ca2þ signals nonetheless have a profound physiological importance for organelle function e.g., vesicle fusion, exocytosis. To record perivesicular Ca2þ, genetically encoded Ca2þ indicators (GECIs) are tethered to the cytosolic face of vesicles as fusion proteins with organellar membrane-associated proteins (tethering also overcomes distortions introduced by the rapid diffusion of mobile Ca2þ indicators). Local Ca2þ has thus been recorded around secretory vesicles (phogrin anchor) (Emmanouilidou et al., 1999; Pouli et al., 1998) and late endosomeelysosomes (TRPML1 or LAMP1 anchors) (McCue, Wardyn, Burgoyne, & Haynes, 2013; Shen et al., 2012).

163

164


FIGURE 3 Organellar cross-talk. (A) The close apposition of different organelle partners, e.g., endolysosomes and the ER proves an intimate architectural framework that allows Ca2þ signals to pass short distances between the two partners, in either direction (Morgan et al. 2013). Traditionally, a small “trigger” release of Ca2þ from the lysosome (e.g., by NAADP) diffuses to the ER where it activates Ca2þ-sensitive channels (e.g., IP3Rs or RyRs). This secondary ER phase of Ca2þ release is usually larger than the endolysosomal release and is thus considered to be the amplification step. (B) Cartoon of a typical agonist-stimulated Ca2þ spike. The multiple phases of Ca2þ release highlight the contribution made by each organelle. The relative proportion and timing of the two components is cell-type dependent and may not be as clearly demarked as this idealized cartoon. (See color plate)

It is worth briefly discussing the Ca2þ affinity of these fusion proteins: to selectively measure local [Ca2þ] around endolysosomal channels (10e100 mM) rather than global [Ca2þ] (nM-mM), the probe must exhibit a low Ca2þ affinity, otherwise the tethered probe simply acts as a cytosolic Ca2þ indicator. To date, the GECIs that have been tethered to vesicles manifest fairly high affinities for Ca2þ (in the sub-tolow micromolar range1), which does not make them ideal for selectively monitoring perivesicular Ca2þ but they could be useful for recording small changes provided that cytosolic Ca2þ signals are accounted for and are different from these local signals. Finally, remember that these probes are still an indirect assessment of vesicular Ca2þ content, albeit a refinement over global cytosolic recordings. In addition, a word of caution: we currently do not know whether pHdso dynamic in the GCaMP3, 0.66 mM Tian, L., Hires, S. A., Mao, T., Huber, D., Chiappe, M. E., Chalasani, S. H. et al. (2009). Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods, 6, 875-881. YC3.6 0.25 mM Palmer, A. E. & Tsien, R. Y. (2006). Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc, 1, 1057e1065, Shen, D., Wang, X., Li, X., Zhang, X., Yao, Z., Dibble, S. et al. (2012). Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nat Commun, 3, 731. 1


lumendfluctuates in the perivesicular domain (which may confound Ca2þ measurements) but this approach demands to be developed and exploited further to assess the role that endolysosomal Ca2þ channels play in organelle biology.

2.3 LUMINAL Ca2D MEASUREMENTS Consequently, the most unambiguous method is to directly monitor the Ca2þ levels solely within the organelle under scrutiny (Figure 2). Recall that the total Ca2þ in the lumen is the sum of the free Ca2þ plus the bound Ca2þ (sequestered by the matrix, usually proteins). Typically, the majority of the luminal Ca2þ is held in reserve in a bound form e.g., in the ER, only 5% is free (Mogami et al., 1999). The first decision to be made is which parameter is more important to the investigator, total or free? The second decision is whether live-cell analysis is essential (as with dynamic changes).

2.3.1 Total Ca2þ The bound/free issue is not often debated by many workers, in part because these two parameters are interrelated: the free Ca2þ is in equilibrium with the bound, so that a fall in the total Ca2þ in the store should be manifested as a fall in the equivalent free [Ca2þ]. However, it is theoretically possible for the total luminal Ca2þ to remain unchanged but, by increased expression of a Ca2þ-binding protein, for example, the free [Ca2þ] would be lowered. Strictly speaking, therefore, both the total and the free Ca2þ should be determined. Total Ca2þ content in acidic vesicles has been assessed by electron microprobe analysis (Morgan et al., 2011) and has been found to be substantial (equating to the millimolar range), as would be expected of a Ca2þ-storing organelle (Morgan et al., 2011). The downside of this approach is that cells are fixed and that organelles may simply be identified by their morphology on the electron micrographs. Plasma mass spectrometry of cell fractions have also been used to assess elemental content (Wang et al., 2012).

2.3.2 Free Ca2þ There are two major methods for determining the free [Ca2þ] inside Ca2þ stores, the second of which we will focus upon in detail.

2.3.2.1 Null-point The first is a null-point technique, which we shall only briefly summarize. In essence, the samples (permeabilized cells or purified vesicles) are immersed in media set at different free [Ca2þ] values and, when the [Ca2þ] in the medium equals the [Ca2þ] in the lumen (the “null point”), there is no further change in a readout parameter (see below). The luminal free [Ca2þ] ([Ca2þ]L) has been thus determined in acidic secretory vesicles (Grinstein, Furuya, Vander Meulen, & Hancock, 1983; Haigh, Parris, & Phillips, 1989) and ER (Dawson, Rich, & Loomis-Husselbee, 1995). In practice, the readouts that have been monitored as a function of the medium [Ca2þ] have been (1) the organelle Ca2þ-leak rate (ER) or (2) pHL (acidic secretory vesicles). Across the ER membrane, the membrane potential (Dj) is close to zero

165

166


and therefore can be ignored, and so the unidirectional leakage of Ca2þ from the organelle occurs predominantly as a function of the Ca2þ concentration gradient across the ER membrane. Note that experiments are performed in the presence of thapsigargin (a SERCA inhibitor, to inhibit Ca2þ reuptake and ensure unidirectional leak). Clearly, when the luminal [Ca2þ] is greater than the medium [Ca2þ], the rate of leak will be fast; when the luminal [Ca2þ] equals the medium [Ca2þ] there is no gradient and so the net rate of leak will be zero (the null-point). Ca2þ leak was monitored with 45Ca2þ when estimating the ER free [Ca2þ] (Dawson et al., 1995). In a different approach, pHL is the readout, usually recorded by a luminal, pH-sensitive fluorescent dye, e.g., acridine orange or endocytosed FITC-dextran in the case of acidic vesicles. In this protocol, a Ca2þ/Hþ-exchanging ionophore (ionomycin or A23187) is used to equilibrate the various bathing-medium Ca2þ concentrations with the Ca2þ of the organelle lumen (Grinstein et al., 1983; Haigh et al., 1989). By virtue of its inherent Ca2þ/Hþ exchange activity, the ionophore will not only translocate Ca2þ but also drive Hþ fluxes, thereby altering the pHL. Hence, when medium [Ca2þ] is higher than that of the lumen, the ionophore facilitates Ca2þ uptake into the organelle lumen at the expense of Hþ loss i.e., the luminal pH (pHL) increases. Conversely, when the medium [Ca2þ] is lower than that of the lumen, the ionophore facilitates Ca2þ egress from the vesicle in exchange for Hþ uptake i.e., pHL decreases. By monitoring pHL, therefore, one can determine when the luminal [Ca2þ] equals the medium [Ca2þ] because there will be no change in pHL (the null point) and the luminal free [Ca2þ] will be determined (Grinstein et al., 1983; Haigh et al., 1989). Pros: only standard equipment (a scintillation counter or fluorimeter). Cons: (1) it requires high protein concentrations of sample; (2) cells are “broken” (either permeabilized or homogenized); (3) there is no single-cell information; (4) one cannot monitor physiological cytosolic Ca2þ signals; (5) it gives no information on luminal Ca2þ dynamics, just a single “time-point”; and (6) Others have questioned the transport assumptions underpinning the relationship between Ca2þ and pH (Erdahl, Chapman, Taylor, & Pfeiffer, 1995).

2.3.2.1 Optical recording 2.3.2.1.1 Background The second strategy for monitoring organellar Ca2þ is optical recording with luminal reporters (Figure 2), which we will discuss in more detail. The advent of Ca2þ-sensitive optical reporters (chemical or genetically encoded) heralded a new phase of Ca2þ signaling research, allowing the noninvasive, direct assessment of [Ca2þ] in intact, living cells. Simply by monitoring the light emitted by these reporters, [Ca2þ] can be monitored and quantified, potentially down to the single cell and subcellular level with high-spatiotemporal resolution. Whether genetically or chemically engineered, these reporters have a common principle of action: the reporter contains Ca2þ-binding motifs (usually carboxylate groups and/or a lone-pair of electrons on nitrogen) that couple to a chromophore; when Ca2þ binds, it alters the electron delocalization (chemical) or conformation (proteins) that changes the light emission from the chromophore motif. Today,


chemical fluorescent reporters such as fura-2 and fluo-3 (or its newer variants) are used routinely to monitor the cytosolic Ca2þ (Takahashi et al., 1999). It was not long before Ca2þ reporters were not only being applied to conventional cytosolic [Ca2þ] determinations but also to measuring [Ca2þ] within organelles themselves. This approach elegantly revealed aspects of organelle biology that could not be otherwise appreciated. Synthetic Ca2þ dyes (fura-2, fluo-4) are usually loaded into cells by incubating cells with the hydrophobic acetoxymethyl ester (AM) precursor; cellular esterases then cleave the AM group, regenerating and trapping the hydrophilic Ca2þ-sensitive molecule in the cell (Takahashi et al., 1999). Usually, the dye loads into the cytosol, but in some cell types it also loads into organelles (for reasons that are not completely understood or controlled) but presumably by virtue of luminal esterases (Takahashi et al., 1999). Although deleterious to cytosolic Ca2þ recording, this dye compartmentation can be advantageously exploited to monitor Ca2þ signals within Ca2þ stores, and has revealed much about dynamics within the ER/SR (Hajnoczky & Thomas, 1997; Launikonis et al., 2006) and mitochondria (Hajnoczky, Robb-Gaspers, Seitz, & Thomas, 1995). Once Ca2þ-reporter proteins were cloned or engineered (e.g., luminescent aequorin or various fluorescent constructs), this offered more precise control over reporter location by judicious introduction of targeting sequences for a broader array of organelles involved in Ca2þ signaling, including mitochondria (Rizzuto, Simpson, Brini, & Pozzan, 1992; Suzuki et al., 2014), ER (Suzuki et al. 2014), Golgi (Lissandron, Podini, Pizzo, & Pozzan, 2010; Pinton, Pozzan, & Rizzuto, 1998), peroxisomes (Drago, Giacomello, Pizzo, & Pozzan, 2008; Lasorsa et al., 2008), and secretory vesicles (Alvarez & Montero, 2002; Dickson, Duman, Moody, Chen, & Hille, 2012; Mitchell et al., 2001; Mitchell et al., 2003; Santodomingo et al., 2008). 2.3.2.1.2 General practical considerations It should be self-evident that organellar reporters need to satisfy a number of criteria, which are as follows: (1) they must be trapped in (or targetted to) the lumen of the organelle of interest; (2) their distribution must be specific to that organelle; (3) their Ca2þ affinity must be appropriate to the range of [Ca2þ] expected in the organelle; (4) ideally, the reporter should be used in a ratiometric (dual wavelength) and not a single wavelength configuration as this eliminates many artifacts and facilitates calibration. Location. The previous section illustrated some general principles of specifically targeting Ca2þ reporters to particular organelles and highlighted the obvious advantages of genetic control over the serendipitous loading of Ca2þ dye AM esters. Affinity. The next and most vital issue is that of Ca2þ affinity (the dissociation constant, Kd), a parameter that impacts upon two aspects of recording Ca2þ. Probably the most obvious issue is that the reporter must operate over the range appropriate to the compartment. Fura-2 has a Ca2þ affinity of w200 nM equating to a working [Ca2þ] range around 20e2000 nM and is therefore appropriate for monitoring cytosolic Ca2þ (Takahashi et al., 1999). Conversely, it would be pointless using it to monitor changes inside Ca2þ stores, which are in the millimolar rangedfura-2 would remain saturated with Ca2þ even if the store was 90% depleted.

167

168


Therefore, investigators have used low-affinity Ca2þ reporters for organellar Ca2þ, be they chemical dyes such as mag-fura-2 (kd 53 mM) and fura-2FF (35 mM) (Hajnoczky & Thomas, 1997) or genetically encoded reporters such as cameleons (Miyawaki, 2011; Palmer & Tsien, 2006) or the newer CEPIAs (w600 mM) (Suzuki et al., 2014). A more subtle issue of the reporter’s Ca2þ affinity is Ca2þ buffering. By definition, all Ca2þ reporters are exogenous Ca2þ buffers and those with a higher Ca2þ affinity will bind (buffer) Ca2þ more avidly than do lower affinity ones. There is, therefore, a balance between having enough reporter to record sufficient light, and having so much that it buffers (and perturbs) the very Ca2þ levels it is trying to measure. In the narrow confines of an organelle lumen this effect could become quite pronounced as reporter levels swamp the endogenous Ca2þ-buffering capacity. In this regard, aequorin is less deleterious than fluorescent reporters because it can be loaded at a concentration that is 2e3 orders of magnitude lower (Pinton, Rimessi, Romagnoli, Prandini, & Rizzuto, 2007). Bottom line: favor the Ca2þ reporter with the lowest useable affinity and use the minimum amount of it. Ratiometric recording. The term refers to dual-color fluorescence recording. Ideally, each channel should show antiparallel changes in fluorescence as a function of Ca2þ; the channel1/channel2 fluorescence ratio is then proportional to the [Ca2þ]. This is typified by the fura-2 family of dyes i.e., a single fluorophore recorded in two channels. Examples of fluorophores showing antiparallel changes are not common, but useable ratios can still be obtained when one of the channels is entirely Ca2þ-insensitive (though the ratio dynamic range is obviously reduced). This is most often encountered in a “pseudo”ratiometric mode where two spectrally distinct fluorophores are used, one being Ca2þ-sensitive and the other Ca2þ-insensitive, e.g., fluo-3 and Texas Red. FRET-based genetically encoded reporters are also used in a ratiometric mode (Miyawaki, 2011). Why is ratiometric recording preferred and more reliable? Because the ratio is far less sensitive to movement, to the fluorophore concentration, fluorophore leakage, and photobleaching2, and simplifies calibration (see below).

3. ASSESSING ENDOLYSOSOMAL Ca2D: SPECIFIC STRATEGIES We will focus upon monitoring Ca2þ with fluorescent indicators. The reader is referred elsewhere for general discussion on the relative merits and pitfalls of cell population or single-cell fluorescence analyses (Cobbold & Rink, 1987). With the appropriate excitation and emission wavelengths for the fluorophore(s), the following strategies can be applied to either cell populations (plate-readers or

2 Photobleaching is entirely corrected for with a single, true ratiometric fluorophore such as fura-2. However, pseudoratiometric recording with two separate fluorophores cannot be absolutely corrected if each fluorophore bleaches at different rates.

3. Assessing Endolysosomal Ca2þ: Specific Strategies

cuvettes) or to single-cell imaging using fluorescence microscopes (epifluorescence or confocal laser scanning).

3.1 INDIRECT MONITORING WITH CYTOSOLIC Ca2D INDICATORS Why and how? Technically and economically, the most appealing method to assess endolysosomal Ca2þ content is to selectively discharge the endolysosomal Ca2þ stores and monitor the appearance of Ca2þ in the cytosol with conventional Ca2þ dyes (Figure 2, Figure 4). When? This technique is merely a qualitative one, originally used as evidence that acidic organelles are Ca2þ-containing stores, e.g., (Churchill et al., 2002; Fasolato et al., 1991). These days, it is best suited to compare the endolysosomal Ca2þ content under different conditions, e.g., in the presence of a drug (Davis et al., 2012), protein expression or disease state (Lloyd-Evans et al., 2008; Shen et al., 2012). N.B. It cannot be “calibrated” and converted into a luminal [Ca2þ].

FIGURE 4 Indirect assaydagents that selectively mobilize acidic Ca2D stores. Upper panels are cartoons depicting the mechanism of action of these different drugs; lower panels are examples of the corresponding cytosolic Ca2þ responses (these examples were from cytotoxic T cells). The dotted lines represent the basal and peak Ca2þ values that should be measured. GPN is cleaved by luminal cathepsin C and results in osmotic swelling and lysosomal membrane rupture that releases Ca2þ. Bafilomycin A1, nigericin and monensin all collapse the pH gradient (alkalinize the lysosome) in different ways, but the neutral pH in each case inhibits Ca2þ uptake and unmasks the Ca2þ leak (in blue). Specifically, bafilomycin A1 blocks the V-Hþ-ATPase and unmasks the Hþ leak; nigericin and monensin are ionophores that directly translocate Hþ from the lumen. NAADP activates Ca2þpermeable channels on the endo-lysosomes (TPCs). (See color plate)

169

170


We shall now detail protocols, pros and cons.

3.1.1 Agents that target acidic Ca2þ stores We currently have a panel of different membrane-permeant drugs that selectively mobilize acidic Ca2þ stores by different mechanisms that rely on different assumptions (Figure 4). We strongly recommend using at least two different classes of agent in order to minimize concerns of side effects or data misinterpretation with one drug alone. 1. GPN (glycyl-L-phenylalanine 2-naphthylamide) is thought to osmotically permeabilize lysosomes and release their luminal Ca2þ. It is a membrane-permeant dipeptide substrate of the lysosomal cysteine (exo)dipeptidase, Cathepsin C (Jadot, Colmant, Wattiaux-De Coninck, & Wattiaux, 1984; Turk, Turk, & Turk, 2001). It does not act upon endosomes, which do not contain Cathepsin C (Berg, Stromhaug, Lovdal, Seglen, & Berg, 1994). a. After GPN cleavage in the lysosome, the luminal products increase the osmotic potential and water uptake results in swelling and breaching of the lysosomal membrane; ions and small MW solutes are released into the cytosol whereas larger molecules (10 kDa) are retained (Haller, Dietl, Deetjen & Volkl 1996; Penny, Kilpatrick, Han, Sneyd, & Patel, 2014). The Ca2þ release we observe occurs through these permeabilization pores (Figure 4). b. The MW cut-off of the pores means that release of lysosomal proteases (>10 kDa) to the cytosol is an unlikely complication. The fact that selective inhibitors of the potentially deleterious Cathepsins B, L and S (but not of Cathepsin C) do not alter GPN responses suggests that this assumption holds (Gerasimenko, Sherwood, Tepikin, Petersen & Gerasimenko 2006; Sanjurjo, Tovey, Prole, & Taylor, 2012; Steen, Kirchberger, & Guse, 2007). c. GPN is reversible (Haller et al., 1996; Pandey et al., 2009). d. We routinely confirm that a given batch (or concentration) of GPN is active by monitoring loss of one of the family of Lysotrackers (Invitrogen), the endolysosomal fluorescent stains. Cells are preloaded with a Lysotracker (e.g., 100 nM, 3 min) and then fluorescence is monitored after addition of DMSO or GPN (Figure 5). e. There are other chemically dissimilar Cathepsin-C substrates that might be used instead of GPN, e.g., methionine-O-methyl ester (SigmaeAldrich), but this is less selective for lysosomes because it also disrupts endosomes (Berg et al., 1994). f. Pros: GPN directly and selectively releases Ca2þ from lysosomes irrespective of ionic circuits (see below); acts rapidly; is reversible. Cons: a crude “shotgun” approach that breaches the lysosomal membrane. 2. Bafilomycin A1 and Concanamycin A are potent inhibitors of the V-Hþ-ATPase that normally acidifies the endolysosomal system (Bowman, Siebers, & Altendorf, 1988; Huss et al., 2002). a. Why does a V-H þ -ATPase inhibitor release Ca2þ? It occurs because of two consecutive consequences of proton pump inhibition, i.e., it releases Ca2þ indirectly (Figure 4).


FIGURE 5 GPN-induced lysis of endolysosomes. Macrophages were loaded with 100 nM Lysotracker Green DND-26 for 3 min and visualized on the stage of a confocal laser-scanning microscope using a standard FITC optical configuration. The fluorescence trace is from a single cell, the inset images show the labeling of a field of cells at different times (indicated in seconds). The addition of 200 mM GPN was indicated and the fall in fluorescence reflects the time course of lysosomal lysis. (See color plate)

b. In brief, the net luminal accumulation of any ion (Hþ or Ca2þ) is a constant battle between uptake and leak; therefore, elimination of uptake unmasks the leak and there is a net loss of ions from the vesicle (Figure 4). The identity of these ion leak pathways is currently uncertain. c. The two consequences of V-Hþ-ATPase inhibition are: (1) the direct unmasking of the Hþ leak that collapses the pH gradient; (2) Ca2þ uptake into endolysosomes depends upon the pH gradient so that Ca2þ uptake is now blocked indirectly; hence, the Ca2þ leak is revealed. In other words, the Ca2þ release stimulated by V-Hþ-ATPase inhibitors is actually the Ca2þ leak. d. For Ca2þ release to occur upon Hþ pump inhibition, the acidic vesicles must be leaky to both Hþ and Ca2þ. However, not all acidic vesicles are leaky, e.g., sea urchin acidic vesicles are not Hþ-leaky and pHL does not change with bafilomycin A1 (Morgan & Galione, 2007b) and they do not release Ca2þ. In T cells, Ca2þ release with bafilomycin A1 is slower and smaller than that with the other acidic store agents, possibly because of slow Hþ leak rates (Davis et al., 2012). Therefore, Hþ pump inhibitors may not work efficiently in every preparation. e. Pros: potent and highly selective for the endolysosomal Hþ pump. Cons: indirect effect on Ca2þ; Ca2þ release can be slow and inefficient depending on the cell-specific ionic circuitry. 3. Nigericin and monensin are ionophores that translocate Hþ across membranes (more correctly, they are electroneutral Kþ/Hþ and Naþ/Hþ exchangers,

171

172


respectively) and therefore directly collapse the endolysosomal pH gradient (Figure 4). Thereafter, it functionally resembles the second stage of Hþ-pump inhibition in that it indirectly inhibits Ca2þ uptake and unmasks the Ca2þ leak. Nigericin/monensin therefore also release Ca2þ via the Ca2þ-leak pathway. a. Pros: rapid effect; does not rely on an endogenous Hþ leak so robustly collapses pH. Cons: indirect effect on Ca2þ; not selective for endolysosomes (translocates Hþ across other membranes including the plasma membrane and other organelles, e.g., mitochondria (Robb-Gaspers et al., 1998)). 4. NAADP is an endogenous second messenger that releases Ca2þ from endolysosomes (Morgan et al., 2011) (Figure 4). It is cell-impermeant but, by analogy with Ca2þ dyes, can be rendered cell-permeant by the masking of its phosphate groups through esterification: the inactive precursor NAADP/AM added outside cells rapidly enters and regenerates active NAADP upon cleavage by intracellular esterases. NAADP/AM is not commercially available but can be synthesized in-house (Parkesh et al., 2008). Because of the delay (seconds to minutes) in attaining a threshold intracellular concentration, the responses are more sluggish compared to the immediacy of GPN etc.

3.1.2 Ca2þ-indicator loading 3.1.2.1 Chemical dyes The reader is referred to any of several practical reviews on the use of chemical Ca2þ dyes (Cobbold & Rink, 1987; Morgan & Thomas, 2006; Simpson, 2006). Suffice to say that for any cell type, it should be empirically determined that the Ca2þ indicator is exclusively loaded into the cytosol (compartmentation of dye into other compartments will result in artifacts). Consequently, we routinely AM-ester load cells at room temperature (which reduces compartmentation). The following describes typical loading with fura-2 (preferred, as it is a ratiometric dyedsee above), but the same principles hold for other Ca2þ dyes. Note that the exact dye concentrations and loading times must be determined for each cell type as they can differ markedly. 3.1.2.1.1 Reagents • •

•

•

3

Fura-2/AM (Invitrogen, Teflabs, EMD Millipore). Stocks 1e5 mM in dry DMSO. Store aliquots several months at 20 C. Keep dark and on ice during day. 10% w/v Pluronic F127 (Invitrogen) in water (initially warm to 37 C to facilitate solubility). Solution can be rewarmed if it later falls out of solution. Store at room temperature several weeks. ECM (extracellular medium), phosphate-free3 extracellular medium (in mM): 121 NaCl, 5.4 KCl, 0.8 MgCl2, 6 NaHCO3, 25 HEPES, 10 Glucose. Prepare with (þ) or without () 1.8 CaCl2. pH 7.4. Add glucose on the day. EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid) (tetrasodium salt; SigmaeAldrich). 0.5 M in water. Neutralize if necessary. Store aliquots 20 C for several months.

The medium must be phosphate-free to avoid Mn2þ precipitating when determining the autofluorescence.


•

Ionomycin (free acid; EMD Millipore or SigmaeAldrich). 1e5 mM in dry DMSO. Store aliquots several months at 20 C. Keep on ice during day. • MnCl2. 2 M stock in water. Store at room temperature for several months. • GPN (Glycyl-L-phenylalanine 2-naphthylamide; Santa Cruz). 200 mM in DMSO. Make up fresh each day from powder, store solution on ice. • Nigericin or monensin (SigmaeAldrich). 10 mM in ethanol. Store aliquots several months at 20 C. Keep on ice during day. • Bafilomycin A1 (Tocris Bioscience). 1 mM in dry DMSO. Store aliquots several months at 20 C. Keep on ice during day. 3.1.2.1.2 Culture and loading 1. Adherent cells are grown to confluency in 96-well plates (for plate reader) or to 70e90% confluency on ethanol-washed glass coverslips (for single-cell imaging). All volumes below are for a 25-mm diameter coverslip in a 35-mm petri dish (or 6-well plate). 2. All ECM solutions are kept at room temperature. 3. Prepare 1 mL of Loading Buffer with 2 mM fura-2/AM (plus 0.03% Pluronic F127, a mild detergent to disperse dye micelles): a. Add 2 mL of fura-2/AM (1 mM stock) to the bottom of an empty microfuge tube. b. Add 3 mL of Pluronic F127 (10% stock) directly onto the fura-2/AM and mix by pipetting. c. Rapidly flood tube with 1 mL of ECM (þ); Vortex. 4. Wash cells once in 1 mL of normal ECM (þ). 5. Replace with 1 mL of Loading Buffer containing the Ca2þ dye. Incubate at room temperature for 45 min, avoiding direct light (dyes are light-sensitive). 6. Remove loading buffer and wash with normal ECM(þ) to remove Ca2þ dye. 7. Incubate for a further 15 min in ECM (þ) to allow complete dye de-esterification. 8. To minimize delays waiting for cell loading, we batch-load several coverslips at the same time. However, we do not leave cells for more than 30e60 min after loading because dyes can slowly leak from cells, even at room temperature.

3.1.2.2 Genetically encoded Ca2þ indicators There are situations when chemical dyes cannot be used: (1) loading is poor or too compartmentalized in a given cell type; (2) a drug of interest interferes with the fluorescence; (3) signal-to-noise is too small. Under these circumstances, we use GECIs, particularly the latest generation orange/red dye, O-GECO1 with a remarkable dynamic range, moderate affinity (minimizing its Ca2þ buffering) and favorable spectrum far from the cellular autofluorescence (Wu et al., 2013). These are expressed as cytosolic proteins. Cells are transfected using conventional lipid-cation transfection reagents such as JetPEI or Lipofectamine and imaged 24 h later.

3.1.3 Ca2þ measurements While monitoring cytosolic Ca2þ, the rationale is to rapidly discharge intracellular Ca2þ stores with agents that selectively target the endolysosomal stores (Figure 4). Runs are brief and best performed in Ca2þ-free medium to eliminate complications

173

174


of Ca2þ influx. Details below are with fura-2 but require minimal modification for other indicators. 1. Remove ECM(þ) and wash cells once in 2 mL of ECM() supplemented with 1e5 mM EGTA to remove Ca2þ. 2. Wash cells twice in ECM() supplemented with lower, 100 mM EGTA (“Ca2þfree medium”) to remove excessive millimolar EGTA. 3. Immediately mount coverslip in a static stage chamber containing 1 mL Ca2þfree medium (þ100 mM EGTA) and image on microscope. 4. For fura-2, record a 350/380 nm image pair every 1e3 s and graph the corresponding ratio against time. 5. Wait a minute or so to establish a quiet, stable baseline and add the agent to discharge the acidic Ca2þ stores (10e200 mM GPN, 1e20 mM nigericin or 0.1e1.0 mM bafilomycin A14). We usually prepare 1 mL of a 2x stock and carefully add this to the chamber and mix5. Vehicle (DMSO) controls should always be performed in parallel. 6. Ca2þ should increase promptly (particularly with GPN or nigericin), attain a peak and then return back to baseline (when in Ca2þ-free medium). Some cells show a simple monotonic rise and fall (Davis et al., 2012; Haller, Volkl, Deetjen & Dietl 1996; Kilpatrick, Eden, Schapira, Futter, & Patel, 2013); others show more complex, oscillatory Ca2þ signals at the single-cell level (Kilpatrick et al., 2013; Penny et al., 2014). The discharge phase should not exceed 10 min in duration. 7. Cellular autofluorescence can be substantial at the fura-2 wavelengths and should be subtracted from the signal post hoc. Note: this is not performed with visiblewavelength dyes. a. Autofluorescence is determined at the end of the run by quenching the fura-2 fluorescence, thereby revealing the residual autofluorescence. b. Fura-2 is quenched by the addition of 2 mM ionomycin plus 2e4 mM MnCl2. Mn2þ quenches fura-2 when it binds; ionomycin facilitates Mn2þ entry. c. Stop the run when the autofluorescence reaches a stable plateau (a few minutes).

3.1.3.1 Analysis 1. If using fura-2, first subtract the autofluorescence. The raw fluorescence of each channel (350 or 380 nm) attains its own fluorescence plateau after Mn2þ The affinity of bafilomycin A1 for the V-Hþ-ATPase is very high (IC50 w1 nM Bowman, E. J., Siebers, A. and Altendorf, K. (1988). Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A, 85, 7972e7976.). We tend to use a high concentration in these release assays so that the kinetics of bafilomycin A1 binding are not limiting i.e., we have to overcome the barriers of the plasma membrane, diffusion, and binding. 5 Note: higher concentrations of GPN (e.g., a 400 mM stock to give 200 mM final) are at the edge of its aqueous solubility and addition of GPN to ECM can result in slow mixing and irreversible precipitation. To circumvent this, we put 2 mL of 200 mM GPN in the bottom of an empty microfuge tube and then add 1 mL of ECM in a rapid action directly to the GPN. Such rapid mixing retains GPN in solution. 4


2. 3.

4.

5.

addition, unique to every single celldsubtract this value from the cell’s previous time points. Plot as the 350/380 ratio, proportional to the intracellular [Ca2þ]. All indicators can be calibrated in terms of absolute [Ca2þ] (Morgan & Thomas, 2006; Simpson, 2006) but there are so many factors that affect Ca2þ reporters in situ (pH, temperature, viscosity, ionic strength, protein binding, other ions) that the calibration is almost rendered invalid and we do not routinely perform this. For monotonic (single-spike) responses, simply measure amplitudes (basal, peak) and kinetics (the initial rate of release) or area-under-the-curve (total amount of Ca2þ) (Figure 4). If cells give complex oscillatory responses, this is more difficult, if not impossible, to interpret in terms of endolysosomal Ca2þ store content (unless there is an obvious difference between treated and controls).

3.1.4 Indirect measurements: pitfalls The ease of execution of these protocols is, unfortunately, offset by the difficulty of interpretation. The Ca2þ signal evoked by GPN etc., is governed not just by the endolysosomal Ca2þ content that we are trying to assess, but also by several other factors inherent to normal Ca2þ homeostasis; ignoring these factors could result in an entirely erroneous interpretation. First, as already alluded to, interorganelle communication is purposely interwoven into the Ca2þ signaling architecture and the biggest problem with the protocols above is that the Ca2þ released from endolysosomes diffuses to and activates adjacent ER Ca2þ channels (IP3Rs and ryanodine receptorsdFigure 3). Such a dialogue is an emerging theme where endolysosomes are the “trigger” that tightly couple to ER, the “amplifier.” Coupling may occur as efficiently with artificial stimuli (GPN, bafilomycin A1, nigericin (Gerasimenko et al., 2006; Kilpatrick et al., 2013)) as it does with a physiological one (NAADP) (Morgan et al., 2011). The upshot of this is that the Ca2þ release one measures with GPN etc., need not be purely from the endolysosomal compartments but may also be “contaminated” with Ca2þ released from the ER (Figure 3). Indeed, as the amplifier, the ER compartment swamps endolysosomes in both volume and Ca2þ content, thus reducing the essential endolysosomal Ca2þ signal component to a minor fraction (Figure 3). Does this actually matter? Strictly speaking, yes it does. For instance, the endolysosomal Ca2þ content may be modestly reduced by a treatment or disease but the change is barely detectable because it is concealed under the ER Ca2þ tidal wave. Of course, how much of a real-world problem this is will depend on the relative fractions of the Ca2þ spike that emanate from endolysosomes and ER, and this is cell-type dependent: the worst (and yet common) case is when the pure endolysosome component is so small that it is undetectable when ER amplification has been eliminated (Cancela, Churchill, & Galione, 1999; Kilpatrick et al., 2013); such cells often manifest Ca2þ oscillations and are difficult to interpret. Conversely, and more favorably, there are cell types where the pure acidic vesicle response

175

176


contributes to greater than 50% of a cytosolic Ca2þ spike (Churchill & Galione 2000), which makes interpretation easier; other cells show an intermediate, detectable endolysosomal contribution (Ruas et al., 2010). Second, any manipulation that alters endolysosomal:ER coupling efficiency (e.g., relative organelle positioning, ER-channel density) or cytosolic Ca2þ buffering (e.g., Ca2þ-ATPase expression) may also impact upon this Indirect assay and result in entirely erroneous conclusions. Additional protocols will be required to test these other possibilities.

3.1.5 Conclusion This is an indirect but technically straightforward assay of endolysosomal Ca2þ content, minimally requiring standard fluorimetric equipment and readily available reagents. However, a sound understanding of the underlying principles of (1) how the Ca2þ-release agents work and (2) the complications arising from interorganelle communication and general Ca2þ homeostasis is absolutely essential: many factors can affect the apparent Ca2þ release in response to GPN etc., and these must be considered and, if needs be, tested. This assay for endolysosomal Ca2þ content can provide a useful indication or an initial screen but it is not definitive and it is only qualitative.

3.2 DIRECT LUMINAL RECORDING In view of the susceptibility of the Indirect method to sundry factors, it is paramount to determine directly the Ca2þ content within the endolysosomal lumen in order to affirm the conclusions drawn from GPN etc. The earlier discussion of general principles of organellar Ca2þ determination in the ER etc., remain relevant (see Optical Recording) but there are additional layers of complexitydsome technical, some biologicaldthat make it difficult (though not impossible) to determine the Ca2þ levels within acidic organelles. We will now highlight the differences and difficulties that these unique vesicles raise and how we might begin to accommodate these vagaries when using luminal Ca2þ reporters.

3.2.1 Luminal pH The most confounding and frustrating issue with measuring the luminal [Ca2þ] in these organelles is their low pHL (whereas it is not an issue for pH-neutral organelles such as the ER). The low pHL (4.5e6.5) potentially impacts upon Ca2þ indicators in two deleterious ways as follows: (1) the chromophore motif may be quenched by acidic pH so that there is insufficient light signal, or (2) the Ca2þ binding motif is pH-sensitive: the Ca2þ-binding site of Ca2þ indicators is often reasonably insensitive to other cations such as Mg2þ, but it does not exclude Hþ which can ably compete with Ca2þ over these pH ranges. The consequence of Hþ competition for the indicator is that it increases the Kd for Ca2þ (i.e., it shifts to a lower Ca2þ affinity), potentially by orders of magnitude i.e., the Kd is pH-dependent (Christensen, Myers, & Swanson, 2002; Martinez-Zaguilan, Parnami, & Martinez, 1998).


3.2.1.1 pH and chromophores This pH-sensitivity of the chromophore is less problematic in the sense that it can be circumvented by choosing a chromophore that is more pH-tolerant i.e., technically, one whose pKa is at least an order of magnitude lower than the pHL of the acidic compartment. For example, the fluorescent protein, EGFP (pKa w6), is quenched pH 7, whereas mCherry is relatively unaffected (pKa pH 6

Oregon green 488 BAPTA-5N Fura-2 dextran

Chem

Fluor

37 mM

Chem

Fluor

Chem

Lysosome

Secretory granule

pKa (Ca2DBinding Site)

Resting [Ca2D]

pH 5.9

w3 mM (15 min) 29 mM (3 min) 3 mM (30 min) 37 mM

w200 mM

pH 4.5

600 mM

Fluor

w500 mM

pH 4.5

400 mM

55.3 mM

Oregon green BAPTA-1 dextran Rhod dextran (low affinity) Calcium orange-5N

Chem

Fluor

690 mM

pH 4.5

550 mM

Chem

Fluor

20 mM*

nd

24 mM

Mag-fura-red

Chem

Fluor

17 mMx

Fluo-4

Chem

Fluor

VAMP2-mut.aequorin{,jj

Gen

Lum

55 mM 10–100 mM

Ca2þ range: 107 –104

pH 5.5-6.3

30–100 mM

References (Christensen et al. 2002) (Gerasimenko, et al. 1998)

(Sherwood et al. 2007) (Christensen et al. 2002) (Christensen et al. 2002) (Lloyd-Evans et al. 2008) (Nguyen et al. 1998; Quesada et al. 2003) (Gerasimenko, O. V. et al. 1996) (Raveh et al. 2012) (Mitchell et al. 2001; Mitchell et al. 2003; Santodomingo et al. 2008)


pH of Ca2D Affinity Assay (and/or pHL)

Chromogranin-aequorin

Gen

Lum

D1-SG

Gen

FRET

Fura-2 (on zymosan particles)

Acrosome

Fura-2 Indo-1 Calcium-green 5N

0.224 mM

Chem

Fluor

nd

w6.2

pH 5.5

1.4 mM

(Mahapatra et al. 2004)

pH-insensitive site

pH 5.8

69 mM

pH 5.0– 8.6

0.58 mM

(Dickson et al. 2012) (LundqvistGustafsson, Gustafsson, & Dahlgren, 2000) (Herrick et al. 2005)

Probe class: Chem (chemical, synthetic), Gen (genetically encoded protein). Optical signal mode: fluor (fluorescence) or lum (luminescence). The Ca2þ affinity (or range) is quoted for the luminal pH of the compartment (see next column). Resting [Ca2þ] in endosomes were determined at the indicated times after dye endocytosis. * Kd does not appear to be pH-corrected. x Unclear whether this Kd was pH-corrected; another group reported a Kd of 55 mM at pH 7.0. Zhao, M., Hollingworth, S. & Baylor, S. M. (1996). Properties of triand tetracarboxylate Ca2þ indicators in frog skeletal muscle fibers. Biophys J, 70, 896-916. { VAMP2 is also known as synaptobrevin2. jj Point mutation in aequorin lowers the Ca2þ affinity. Montero, M., Brini, M., Marsault, R., Alvarez, J., Sitia, R., Pozzan, T. et al. (1995). Monitoring dynamic changes in free Ca2þ concentration in the endoplasmic reticulum of intact cells. EMBO J, 14, 5467–5475. # Similar Ca2þ range to cytosolic aequorin. ** Dominant low-affinity site.


Phagosome

Ca2þ range#: 10 7 –105 60 mM**

179

180


Recently, a luminal FRET-based probe was engineered that targets the lumen of acidic secretory granules (pH 5.5) which, remarkably, showed little change in Ca2þ affinity between pH 7.4 and 5.5 (Dickson et al., 2012). The new D1-SG probe was based around a low affinity cameleon (D1-ER) with modified calmodulin and myosin light-chain kinase peptide motifs (Palmer, Jin, Reed, & Tsien, 2004). Although, the pKa values of these mutated Ca2þ-binding peptides have not been published (to the best of our knowledge), this may indeed represent a promising new pH-insensitive template for other acidic organelles.

3.3 LUMINAL RECORDING: PRACTICALITIES To date, the majority of dynamic acidic organelle Ca2þ measurements have been performed in secretory vesicles but other stores have also been studied, albeit to a lesser degree, and include endosomes, late endosomeelysosomes, and phagosomes (Table 1). We shall discuss strategies and practicalities for each step of the protocol.

3.3.1 Targeting indicators to acidic vesicles 3.3.1.1 Chemical indicators: ester As mentioned above, rapid (60 min) AM ester-loading of the cells can result in Ca2þ dyes compartmentalizing into organelles, and this has been successfully used to load and monitor Ca2þ within the lumen of secretory vesicles. For the most part, this has been carried out in purified or enriched granule preparations because the cytosol is also labeled and would contaminate the vesicular signal (Gerasimenko, Gerasimenko, Belan & Petersen 1996; Nguyen, Chin, & Verdugo, 1998; Quesada et al., 2003; Raveh et al., 2012). The inference is that secretory vesicles contain esterases and retain the dyes. In contrast, reports of AM-loading of endolysosomes are sparse (Herrick et al., 2005; Trollinger, Cascio, & Lemasters, 2000), which implies that they lack sufficient esterase activity and/or possess transporters that eject the dyes from the lumen. Not the method of choice for endolysosomes.

3.3.1.2 Chemical indicators: endocytosis An established strategy for labeling the lumen of endolysosomes is to bathe cells in medium containing the indicator which is then taken up via endocytosis (Figure 6(A)). From these nascent and early endosomes, the dye will then traffic through to late endosomes and, eventually, lysosomes. Therefore, one can selectively label compartments of this pathway by incubating cells with the dye for appropriate times (see below), e.g., short times (min to hours) for endosomes, longer times (overnight) for lysosomes. What form of the reporter should be used for endocytotic loading? Not an esterified precursor but rather the hydrophilic Ca2þ-binding (salt) form. However, as already alluded to, acidic organelles have the capacity to eject (transport) small MW dye molecules from their lumina with some efficiency; this means that the small Ca2þ dyes would not be retained for long, and can only realistically monitor

(A) Strategy for labeling lysosomes by incubating with extracellular dextrans; endosomal labeling is later “chased” out by incubating in media without dextrans. (B) To determine which compartment endocytosed dextrans are located in, cells loaded overnight with fixable Texas Red Dextran are fixed and then immunolabeled with antibodies against organelle markers such as EEA1 and LAMP1. Dextran overlaps poorly with the early endosome marker but shows a high correlation with the late-endosome/lysosome marker, LAMP1. Scale bar ¼ 10 mm. (C) In vitro determination of the Kd for Ca2þ for a Ca2þ indicator at neutral and acidic pH. This example is for low-affinity rhod dextran at pH 7.2 and pH 4.5. The free [Ca2þ] was clamped using different concentration ratios of total Ca2þ:Ca2þ chelators. (D) Cartoon of quasi-ratiometric recording of luminal Ca2þ changes in response to NAADP assessed with rhod and Alexa Fluor 488 dextrans. (See color plate)


FIGURE 6 Direct monitoring of the luminal Ca2D.

181

182


Ca2þ in early compartments (endosomes) that require short loading times (Gerasimenko, Tepikin, Petersen & Gerasimenko 1998; Sherwood et al., 2007). For later compartments, it is essential to prevent dye extrusion by conjugating the dye to a large MW carrier (therefore no longer a transporter substrate). Thus, Ca2þ dyes conjugated to proteins, beads or dextrans survive in the endolysosomal system and allow Ca2þ recording. Although an expensive protocol because of the amount of extracellular dye required, it is a feasible one for critical experiments (and one that can be adapted for monitoring pHL).

3.3.1.3 Genetic indicators To date, GECIs have only been targeted to secretory vesicles, by generating chimeras with vesicle proteins such as chromogranin A (Mahapatra et al., 2004), VAMP2 (Mitchell et al., 2001, 2003; Santodomingo et al., 2008), and tissue plasminogen activator (Dickson et al., 2012). It is unclear whether similar strategies could be exploited for endolysosomes; irrespective of the pHL issues raised above, the aggressive environment of the endolysosomal lumen (pHL and enzymatic hydrolysis) can result in the instability and degradation of heterologously expressed proteins (Pryor, 2012). That is, even if fusion proteins could be generated and correctly targeted to the lumen, would these indicators survive in this environment? The field is crying out for the rationale design of genetically encoded luminal [Ca2þ] indicators for endolysosomes but these will have to account for the pHL sensitivity as well as the stability of the protein itself and, unfortunately, this currently seems a long way off.

3.3.2 Resting or dynamic [Ca2þ] changes? Having chosen the Ca2þ indicator and loading protocol, the next issues relate to the biological question being posed: Is the requirement for simply assessing the “resting” luminal [Ca2þ] or for recording acute, dynamic Ca2þ changes, e.g., during cell stimulation? Given that pHL interferes with the Ca2þ indicator, it is far more straightforward to record the resting [Ca2þ] values (when pHL is likely to be stable). On the other hand, dynamic Ca2þ changes may be accompanied by dynamic pHL changes (see above), which makes for a more difficult interpretation of indicator signal fluctuations. We find the resting values more reliable but accept that dynamic recording may be feasible with appropriate controls (see below).

3.3.3 Calibration and correcting for pHL The advantage of direct recording is that one can, in theory, obtain an absolute value for the free [Ca2þ] in the endolysosomal lumen, thus dispelling the uncertainties surrounding the indirect approach. Calibrating the luminal Ca2þ dye in terms of absolute [Ca2þ] does, however, require several additional practical steps.

3.3.3.1 pHL correction

Calibration requires an accurate Kd of the dye for Ca2þ, but the sensitivity of the Kd to Hþ means that (1) the resting pHL of the compartment must be accurately determined and (2) the Kd of the indicator must be determined at that pH.


We use standard techniques to determine pHL of the appropriate endolysosomal compartment(s) using endocytosed dextran-conjugated pH dyes (FITC or Oregon Green 488) in a ratiometric mode and calibrated with high Kþ media and Kþ/Hþ ionophores (nigericin and valinomycin) (see Grinstein et al. (Chapter 5 of this volume)) (Christensen et al., 2002; Lloyd-Evans et al., 2008). Determining the Kd of the Ca2þ dye at a given pH is, by necessity, conducted in vitro because it requires a strict control over pH and [Ca2þ]. In brief, prepare solutions across a large range of [Ca2þ] and monitor the indicator fluorescence (or luminescence) as a function of [Ca2þ]. We recommend recording the Ca2þ affinity at two pH values, one close to neutrality and one at the acidic pH (Figure 6(C)). The former is a positive control to allow direct comparison with the published Kd around neutral pH, thereby giving more confidence that the assay is working. The downside of an in vitro determination of Ca2þ affinity is that, by definition, the assay conditions will not precisely emulate the environment of the organelle lumen; we noted above that several biophysical factors affect the Kd and so the dye may not manifest the same affinity in situ as in vitro. This is slightly unsatisfactory and means that absolute luminal [Ca2þ] must be treated with a degree of latitude, but it is really the only option open to us (note: calibrating in situ with Ca2þ ionophores is not possible because they act as Ca2þ/Hþ exchangers and will not act upon acidic vesicles without changing pHL). The better news is that any slight uncertainty surrounding absolute [Ca2þ] values will not affect conclusions about the relative alteration in the resting [Ca2þ] (e.g., as a result of disease), provided that the pHL is unaffected; that is, the Kd is used to calculate the [Ca2þ] for all treatments and if it is twofold in error, then the [Ca2þ] will be twofold in error for all treatmentsdall treatments will be equally scaled up or down.

3.3.3.2 Ratiometric recording A prerequisite for calibrating luminal fluorescent Ca2þ indicators in living cells is recording in a ratiometric mode i.e., dual color. Other workers have successfully used a true ratiometric dye, fura-2 dextran (Christensen et al., 2002). This is still commercially available from Invitrogen but is expensive. Alternatively, we use a pseudoratio protocol with two dyes (Figure 6(D)), coloading endolysosomes with a Ca2þ-sensitive dye plus a different color Ca2þ-insensitive one (analogous to pH protocols (Lloyd-Evans et al., 2008)); needless to say, neither fluorophore must be pH-sensitive. The ratio of these two signals is proportional to [Ca2þ]; one need only know the Kd, and the ratios in the absence of Ca2þ (Rmin) and presence of saturating Ca2þ (Rmax) to convert the ratio into an absolute [Ca2þ] (see below). Rmin and Rmax have either been determined in live cells in situ (Christensen et al., 2002; Sherwood et al., 2007) or in vitro (Lloyd-Evans et al., 2008), each with its pros and cons. In situ. Uses the Ca2þ ionophore, ionomycin, to equilibrate Ca2þ across the organelle membrane. Pros: relatively rapid; post hoc in the same cells as the experiment was conducted; the dye is calibrated within the organelle. Cons: the luminal indicator is calibrated at neutral pH (after addition of nigericin) whereas recordings were at physiological acidic pH; although Rmin and Rmax can then be determined by

183

184


Ca2þ ionophore (ionomycin), there are occasions when ionomycin cannot fully equilibrate cellular Ca2þ with extracellular Ca2þ because endogenous Ca2þ pumps buffer the imposed changes (Thomas et al., 2000); this results in an underestimate of the dynamic range and, thereby a false [Ca2þ] value. In vitro. Record fluorescence in just two solutions prepared with the extremes of Ca2þ. Pros: reliable manipulation of the Ca2þ levels bound to the indicator, thereby obtaining Rmin and Rmax at acidic pH. Cons: dye not in authentic luminal environment. We favor this approach because we find it more reliable to obtain credible Rmin and Rmax values (and there is probably a larger error from incorrect Rmin and Rmax than from other more subtle biophysical effects).

3.3.4 Luminal Ca2þ protocol 3.3.4.1 Reagents • • • • • • • • • • •

• •

6

Low-affinity6 rhod dextran (10 kDa, custom synthesis, discontinued by Invitrogen). Stock solution 5 mg/mL in tissue culture medium. Store aliquots 20 C. Alexa Fluor 488 dextran (Invitrogen). Stock solution 5 mg/mL in tissue culture medium. Store aliquots 20 C. Fura-dextran (Invitrogen). Stock solution 5 mg/mL in tissue culture medium. Store aliquots 20 C. Texas Red Dextran (10 kDa, fixable, Invitrogen, D-1863). Stock solution 5 mg/ mL in tissue culture medium. Store aliquots 20 C. BAPTA (tetra-sodium salt; SigmaeAldrich). 0.5 M in water. Store aliquots 20 C for several months. 5,5’-dibromo-BAPTA (Br2-BAPTA; tetra-potassium salt; Invitrogen). 0.5 M in water. Store aliquots 20 C for several months. CaCl2. 1 M, analytical grade. Neutral intralysosomal medium (ILM, mM): 10 NaCl, 140 KCl, 1 MgCl2, 5 BAPTA (or Br2-BAPTA), 7 Hepes (acid), 3 Hepes (Naþ salt), pH 7.2. Acidic intralysosomal medium (ILM, mM): 10 NaCl, 140 KCl, 1 MgCl2, 5 BAPTA (or Br2-BAPTA), 6 acetic acid, 4 sodium acetate, pH 4.5. Rmin solution: ILM only. Rmax solution: ILM þ 5 mM CaCl2. The pH of this solution must be readjusted (e.g., with KOH) because Ca2þ displacement of Hþ from BAPTAs will significantly acidify the ILM. ECM, extracellular medium (in mM): 121 NaCl, 5.4 KCl, 0.8 MgCl2, 6 NaHCO3, 25 HEPES, 10 Glucose, 1.8 CaCl2, pH 7.4. Borosilicate capillary glass with an internal filament (as used for pulling microelectrode or injection pipettes), e.g., 0.78 mm ID, 1.00 mm OD (Harvard Apparatus).

Do not use the high-affinity version as this avidly chelates luminal Ca2þ and induces a pathological phenotype Lloyd-Evans, E., Morgan, A. J., He, X., Smith, D. A., Elliot-Smith, E., Sillence, D. J. et al. (2008). Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat Med, 14, 1247e1255.


3.3.4.2 In vitro determination of the Kd of the Ca2þ dye 3.3.4.2.1 General points • The dye’s Ca2þ affinity is independent of the hardware, so it is not essential to determine this on the microscope (a cumbersome process). We use a cuvettebased fluorimeter or, more usually, a fluorescence plate reader (that uses less reagents and is faster). • To construct a Ca2þ calibration curve with solutions at different free [Ca2þ], we clamp the medium Ca2þ to precalculated values using different ratios of Chelator and Ca$Chelator media (Table 2). Because of the exquisite pHsensitivity of EGTA, we prefer the reliability of BAPTA and Br2-BAPTA (Ca2þ Kd at neutral pH: 0.11 mM and 1.6 mM, respectively) (Tsien, 1980). For different chelator/Ca2þ ratios, the free [Ca2þ] is calculated using calculators such as Chris Patton’s Maxchelator (http://maxchelator.stanford.edu/webmaxc/ webmaxcE.htm). See Table 2 for formulation. • We clamp the pH of solutions with buffers with a pKa appropriate for the pH range required (pKa at 20 C: Hepes, 7.55; MES, 6.4; acetate 4.74). All pH and Ca2þ Table 2 Buffer Composition to Clamp the Free [Ca2þ] at Different Values Neutral pH 7.2 BAPTA

Acidic pH 4.5 Br2-BAPTA

BAPTA

Br2-BAPTA

2D

Total Ca added (mM)*

[Ca2D]free (mM)

0x 1 1.5 2 2.5 3 3.5 4 4.5

0.0002 0.0632 0.108 0.169 0.253 0.38 0.591 1.01 2.2

0.002 0.57 0.98 1.5 2.2 3.4 5.2 9.0 19.6

0.17 41.4 70.0 107 155 219 309 435 612

1 10 50

-

-

-

0.0587 14.5 24.8 38.3 56.7 83.1 124 190 308 Without chelator 1000 10,000 50,000

An intralysosomal-like medium is buffered to pH 7.2 with Hepes or to 4.5 with acetate. Media at these pH values are supplemented with 5 mM Ca2þ chelator (BAPTA or the lower affinity Br2-BAPTA). Increasing the total amount of Ca2þ added results in the free [Ca2þ] indicated. No single Ca2þ chelator covers the entire range required for low-affinity rhod dextran (Kd w5 mM at neutral pH) and so we are obliged to straddle their two ranges (in which case we overlap the ranges to ensure there is no artifact of using a different chelator). * For acidic media, the three highest free [Ca2þ] are in the absence of Br2-BAPTA. x Assume that total Ca2þ contamination is 5 mM.

185

186


•

calculations assume room temperature (20 C). Our example determines the Kd at pH 4.5, but this value depends upon the pHL of your compartment of interest. In vitro, it is not absolutely necessary to record the Ca2þ/fluorescence relationship ratiometrically, single wavelength (red) recording will suffice. However, it is therefore advisable to minimize interwell variations in the dye concentration from pipetting errors; hence, for each pH, prepare only two stock media (e.g., BAPTA, CaBAPTA) and mix these in different volume ratios to obtain different free [Ca2þ].

3.3.4.2.2 Protocol Prepare minimum volumes appropriate for semimicro cuvettes or multiwell plates. 1. It is convenient to prepare a common ILM without pH-buffers or Ca2þ-buffers. 2. Using the HendersoneHasselbalch equation, add appropriate volumes of acid/ base forms of Hepes (or acetate) from 1 M stocks to give two solutions at each required neutral or acidic pH. 3. Supplement these solutions with chelators (Ca2þ) to give eight solutions, all pH-adjusted (especially those with Ca2þ): 4. To each solution, add 5 mM rhod dextran. Neutral

5 mM BAPTA

–Ca2D

DCa2D

•

•

5 mM Br2BAPTA 5 mM Ca2þ

Acidic

–Ca2D

•

•

DCa2D

–Ca2D

DCa2D

•

•

•

•

•

–Ca2D

DCa2D

•

•

•

5. Mix solution pairs (Ca2þ) in different volume ratios (Table 2) to obtain the required different total [Ca2þ]. Thus, Ca2þ chelator and indicator concentrations will be constant. Note: different free [Ca2þ], ionic strength and/or different pH will require recalculation with Maxchelator. 6. At acidic pH, the highest free Ca2þ concentrations can only be obtained at acidic pH by adding Ca2þ without Br2-BAPTA. 7. At room temperature record the fluorescence at each [Ca2þ] in a fluorimeter or plate-reader using rhodamine excitation/emission wavelengths, e.g., 550/580 nm. 8. Plot the fluorescence versus free [Ca2þ] and determine each Kd by standard single-binding site curve fitting, e.g., in Graphpad Prism software (Figure 6(C)).

3.3.4.3 Cell loading with dyes by endocytosis 1. For live-cell imaging, adherent cells are seeded onto glass coverslips at least one day prior to dye loading. Use as small a coverslip and chamber as possible to minimize prohibitive expense of high-dextran concentrations.


2. Cells are incubated overnight (w16 h) in culture medium supplemented with a Ca2þ-sensitive dye, e.g., low-affinity rhod dextran plus a Ca2þ- (and pH-) insensitive dye (Alexa Fluor 488 dextran). Ensure to use the minimum concentration of dyes that allows a good signal-to-noise recording without causing swelling and fusion of endolysosomes (Pryor, 2012). A typical range is 0.1e1.0 mg/mL. Note: the two dyes need not be equimolar (but once a ratio is selected, this should be used throughout the protocol, in vitro or in situ). 3. Next day, the medium containing the excess dye that has not been endocytosed is carefully removed and can be stored sterile and reused up to 2x more on other cells in the interests of economy. 4. To specifically label later compartments (lysosomes), chase the endocytosed dyes from the endosomes by incubating cells for a further in fresh tissue culture medium without dyes (Figure 6(A)). For fibroblasts, cells are washed 3x and then incubated in dextran-free medium for a further 8 h. Other cell types are labeled for 5 h and then chased overnight (empirically determined). 5. Note: for each cell type, we first verify which compartments the dextrans label under the incubation conditions by using lysine-fixable dextrans (e.g., 0.2 mg/ mL fixable Texas-Red dextran). After fixation in 4% paraformaldehyde, and by confocal laser scanning microscopy, we compare the dextran colocalization with immunolabeling of standard acidic organelle markers (Figure 6(B)), e.g., transferrin receptor (recycling endosomes) EEA1 (early endosomes), LAMP1 (endolysosomes), Rab7 and (late endosomes). Quantify colocalization with Pearson or Manders coefficients.

3.3.4.4 Imaging Ca2þ indicator fluorescencedcells 1. Cells are mounted in a stage chamber in ECM. 2. Capture images using standard FITC and rhodamine filter sets. For example, excitation/emission (nm): 488/505e530 (green), 543/>560 (red). 3. N.B. It is absolutely essential that all acquisition settings (e.g., light intensity, detector sensitivity, image resolution, channel configuration) are identical for all cell treatments and calibration steps. They cannot be directly compared otherwise. 4. Tip: first ensure that the acquisition settings allow the calibration limits (especially the raw red Ca2þ-dye fluorescence at Rmax) to remain on-scale and do not saturate the detector. There is no point in recording the cells at the brightest possible settings if the raw Ca2þ-dye fluorescence subsequently exceeds the detector capacity at saturating Ca2þ (meaning that Rmax cannot be determined and that the cell fluorescence cannot be calibrated).

3.3.4.5 Imaging Ca2þ indicator fluorescence e calibration

To convert the lysosomal fluorescence into [Ca2þ] we use the following standard equation, as originally reported for fura-2 (Grynkiewicz, Poenie, & Tsien, 1985):

187

188


2þ Sf 2 ðR Rmin Þ Ca ¼ Kd Sb2 ðRmax RÞ where R is the fluorescence ratio of rhod/Alexa Fluor 488 dextrans. Rmin represents the ratio in the absence of Ca2þ whereas Rmax is the ratio in the presence of a saturating [Ca2þ]. Sf2 and Sb2 are the fluorescence of the denominator (Alexa Fluor 488) in the absence and presence of Ca2þ, respectively7. The units of the [Ca2þ] are the same as those used for the Ca2þ dissociation constant (Kd) (usually nM or mM). 3.3.4.5.1 Protocol 1. The Kd was determined in vitro (see above). 2. Rmin and Rmax must be determined on the microscope stage with the microscope acquisition parameters identical to those used for the cells. 3. Record the fluorescence of ILM solutions containing rhod dextran and Alexa Fluor 488 dextran in the same ratio used for loading cells8. Practically, to minimize wastage of expensive dyes, we deliver the solutions to the microscope stage in as small a volume as feasible that avoids evaporation. We have tested two different strategies with essentially the same results that are as follows: a. Gently apply 50e100 mL as a concentrated drop to the center of an empty, dry cell chamber (with a clean coverslip as the base). b. Alternatively, break capillary glass into small sections, w1 cm in length, and partially fill them with a few ml of a solution using a standard hand pipette (narrow-gauge pipette tips can be advantageous). Place them into an empty cell chamber as above. Use different capillaries for Rmin and Rmax. 4. At acidic pH, the Rmax solution must be saturated with Ca2þ; in our example, we omitted chelators and added 50 mM Ca2þ (Table 2). 5. Record 4e6 different images in different regions of the drop or capillary for each parameter. Avoid edge artifacts. Record the raw green/red fluorescence in each region and mean the Rmin or Rmax values.

3.3.4.6 Dynamic luminal Ca2þ changes Rather than static, basal [Ca2þ] recordings, the same methodology could, in theory, be applied to monitoring endolysosomal luminal [Ca2þ] fluctuations during cell stimulation; this would be wonderfully useful to understand more about how endolysosomal Ca2þ channels and transporters are regulated during complex signaling. As discussed earlier, however, the current Ca2þ indicators’ sensitivity to pH makes such recordings less reliable (particularly when pHL dynamically fluctuates at the same time). Of course, simultaneous (or at least parallel) recordings of pHL can 7

With fura-2 these values are Ca2þ-dependent and are usually a high ratio (5e15). With the pseudoratio mode, Alexa Fluor 488 should be Ca2þ-independent and therefore this expression will be w1, and can usually be ignored. 8 Note: it is not necessary (or indeed advisable) to use these dextrans at the same absolute concentrations as used for cell loading; dilute them equally so that the absolute fluorescence is in a range comparable to the cell fluorescence.

References

be used to correct (or account) for alterations of the Ca2þ Kd (Santodomingo et al., 2008) or chromophore fluorescence (Dickson et al., 2012), but the calculations and assumptions are not straightforward and no substitute for the confidence that a pH-insensitive Ca2þ indicator would engender. We would caution against applying this technique until such concerns are engineered away.

3.3.5 Conclusions Pros: the only method for quantifying the absolute free [Ca2þ] within the organelles; not confounded by cytosolic Ca2þ complexities and uncertainties of ion homeostasis; live-cell recording. Cons: pH-sensitivity; multiple steps required (determination of compartment pHL and Ca2þ-dye Kd at pHL; live-cell endolysosomal recording); too expensive for routine assays; scarce commercial availability of dextranconjugates may necessitate custom synthesis.

4. FINAL REMARKS The convergence of the fields of Ca2þ signaling and endolysosomal biology ensures that the interest in measuring the Ca2þ content of (and Ca2þ release from) acidic organelles will only increase and, in all likelihood, dramatically. As this chapter illustrates, the current techniques available to us are far from optimal and not a little daunting for the nonexpert. Nonetheless we are hopeful that, with care, these methodologies can prove valuable to fields as diverse as autophagy, lysosomal storage diseases, trafficking, and cell signaling. Whenever possible, the Direct method is the one of choice, but it is not easy (or cheap) to implement and the Indirect approach will probably prove the more popular on technical grounds alone. We would, therefore, encourage investigators to be mindful of the cautionary notes we raise before (potentially) overinterpreting data generated by the Indirect method. The field will warmly welcome the development of pH-insensitive genetically encoded luminal Ca2þ reporters so that this becomes a more routine approach, available to all.

REFERENCES Aley, P. K., Mikolajczyk, A. M., Munz, B., Churchill, G. C., Galione, A., & Berger, F. (2010). Nicotinic acid adenine dinucleotide phosphate regulates skeletal muscle differentiation via action at two-pore channels. Proceedings of the National Academy of Sciences of the United States of America, 107, 19927e19932. Alvarez, J., & Montero, M. (2002). Measuring [Ca2þ] in the endoplasmic reticulum with aequorin. Cell Calcium, 32, 251e260. Berg, T. O., Stromhaug, E., Lovdal, T., Seglen, O., & Berg, T. (1994). Use of glycylL-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles. Biochemistry Journal, 300(Pt 1), 229e236. Berridge, M. J. (2009). Inositol trisphosphate and calcium signalling mechanisms. Biochimica et Biophysica Acta, 1793, 933e940.

189

190


Berridge, M. J., Bootman, M. D., & Roderick, H. L. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nature Reviews Molecular Cell Biology, 4, 517e529. Bowman, E. J., Siebers, A., & Altendorf, K. (1988). Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proceedings of the National Academy of Sciences of the United States of America, 85, 7972e7976. Brailoiu, G. C., Brailoiu, E., Parkesh, R., Galione, A., Churchill, G. C., Patel, S., et al. (2009b). NAADP-mediated channel ’chatter’ in neurons of the rat medulla oblongata. Biochemistry Journal, 419, 91e97. Brailoiu, E., Churamani, D., Cai, X., Schrlau, M. G., Brailoiu, G. C., Gao, X., et al. (2009a). Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling. Journal of Cell Biology, 186, 201e209. Brailoiu, E., Churamani, D., Pandey, V., Brailoiu, G. C., Tuluc, F., Patel, S., et al. (2006). Messenger-specific role for nicotinic acid adenine dinucleotide phosphate in neuronal differentiation. Journal of Biological Chemistry, 281, 15923e15928. Calcraft, P. J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., et al. (2009). NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature, 459, 596e600. Campbell, T. N., & Choy, F. Y. M. (2001). The effect of pH on Green fluorescent protein: a brief review. Molecular Biology Today, 2, 1e4. Cancela, J. M., Churchill, G. C., & Galione, A. (1999). Coordination of agonist-induced Ca2þsignalling patterns by NAADP in pancreatic acinar cells. Nature, 398, 74e76. Christensen, K. A., Myers, J. T., & Swanson, J. A. (2002). pH-dependent regulation of lysosomal calcium in macrophages. Journal of Cell Science, 115, 599e607. Churchill, G. C., & Galione, A. (2000). Spatial control of Ca2þ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients. Journal of Biological Chemistry, 275, 38687e38692. Churchill, G. C., Okada, Y., Thomas, J. M., Genazzani, A. A., Patel, S., & Galione, A. (2002). NAADP mobilizes Ca2þ from reserve granules, a lysosome-related organelle, in sea urchin eggs. Cell, 111, 703e708. Cobbold, P. H., & Rink, T. J. (1987). Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochemistry Journal, 248, 313e328. Coen, K., Flannagan, R. S., Baron, S., Carraro-Lacroix, L. R., Wang, D., Vermeire, W., et al. (2012). Lysosomal calcium homeostasis defects, not proton pump defects, cause endolysosomal dysfunction in PSEN-deficient cells. Journal of Cell Biology, 198, 23e35. Collins, T. P., Bayliss, R., Churchill, G. C., Galione, A., & Terrar, D. A. (2011). NAADP influences excitation-contraction coupling by releasing calcium from lysosomes in atrial myocytes. Cell Calcium, 50, 449e458. Cosker, F., Cheviron, N., Yamasaki, M., Menteyne, A., Lund, F. E., Moutin, M. J., et al. (2010). The ecto-enzyme CD38 is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2þ mobilization from lysosomes in pancreatic acinar cells. Journal of Biological Chemistry, 285, 38251e38259. Davis, L. C., Morgan, A. J., Chen, J. L., Snead, C. M., Bloor-Young, D., Shenderov, E., et al. (2012). NAADP activates two-pore Channels on T Cell Cytolytic granules to stimulate exocytosis and Killing. Current Biology, 22, 1e7. Davis, L. C., Morgan, A. J., Ruas, M., Wong, J. L., Graeff, R. M., Poustka, A. J., et al. (2008). Ca2þ signaling occurs via second messenger release from intraorganelle synthesis sites. Current Biology, 18, 1612e1618.

References

Dawson, A. P., Rich, G. T., & Loomis-Husselbee, J. W. (1995). Estimation of the free [Ca2þ] gradient across endoplasmic reticulum membranes by a null-point method. Biochemistry Journal, 310(Pt 2), 371e374. Dickson, E. J., Duman, J. G., Moody, M. W., Chen, L., & Hille, B. (2012). OraiSTIM-mediated Ca2þ release from secretory granules revealed by a targeted Ca2þ and pH probe. Proceedings of the National Academy of Sciences of the United States of America, 109, E3539eE3548. Dong, X.-P., Shen, D., Wang, X., Dawson, T., Li, X., Zhang, Q., et al. (2010). PI(3,5)P2 controls membrane trafficking by direct activation of mucolipin Ca2þ release channels in the endolysosome. Nature Communications, 1, 1e11. Drago, I., Giacomello, M., Pizzo, P., & Pozzan, T. (2008). Calcium dynamics in the peroxisomal lumen of living cells. Journal of Biological Chemistry, 283, 14384e14390. Emmanouilidou, E., Teschemacher, A. G., Pouli, A. E., Nicholls, L. I., Seward, E. P., & Rutter, G. A. (1999). Imaging Ca2þ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon. Current Biology, 9, 915e918. Erdahl, W. L., Chapman, C. J., Taylor, R. W., & Pfeiffer, D. R. (1995). Effects of pH conditions on Ca2þ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophysical Journal, 69, 2350e2363. Fasolato, C., Zottini, M., Clementi, E., Zacchetti, D., Meldolesi, J., & Pozzan, T. (1991). Intracellular Ca2þ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca2þ accumulation, storage, and release. Journal of Biological Chemistry, 266, 20159e20167. Gerasimenko, O. V., Gerasimenko, J. V., Belan, P. V., & Petersen, O. H. (1996). Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2þ from single isolated pancreatic zymogen granules. Cell, 84, 473e480. Gerasimenko, J. V., Sherwood, M., Tepikin, A. V., Petersen, O. H., & Gerasimenko, O. V. (2006). NAADP, cADPR and IP3 all release Ca2þ from the endoplasmic reticulum and an acidic store in the secretory granule area. Journal of Cell Science, 119, 226e238. Gerasimenko, J. V., Tepikin, A. V., Petersen, O. H., & Gerasimenko, O. V. (1998). Calcium uptake via endocytosis with rapid release from acidifying endosomes. Current Biology, 8, 1335e1338. Grinstein, S., Furuya, W., Vander Meulen, J., & Hancock, R. G. (1983). The total and free concentrations of Ca2þ and Mg2þ inside platelet secretory granules. Measurements employing a novel double null point technique. Journal of Biological Chemistry, 258, 14774e14777. Grynkiewicz, G., Poenie, M., & Tsien, R. Y. (1985). A new generation of Ca2þ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry, 260, 3440e3450. Haigh, J. R., Parris, R., & Phillips, J. H. (1989). Free concentrations of sodium, potassium and calcium in chromaffin granules. Biochemistry Journal, 259, 485e491. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., & Thomas, A. P. (1995). Decoding of cytosolic calcium oscillations in the mitochondria. Cell, 82, 415e424. Hajnoczky, G., & Thomas, A. P. (1997). Minimal requirements for calcium oscillations driven by the IP3 receptor. Embo Journal, 16, 3533e3543. Haller, T., Dietl, P., Deetjen, P., & Volkl, H. (1996a). The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2þ release. Cell Calcium, 19, 157e165.

191

192


Haller, T., Volkl, H., Deetjen, P., & Dietl, P. (1996b). The lysosomal Ca2þ pool in MDCK cells can be released by ins(1,4,5)P3- dependent hormones or thapsigargin but does not activate store-operated Ca2þ entry. Biochemistry Journal, 319, 909e912. Herrick, S. B., Schweissinger, D. L., Kim, S. W., Bayan, K. R., Mann, S., & Cardullo, R. A. (2005). The acrosomal vesicle of mouse sperm is a calcium store. Journal of Cellular Physiology, 202, 663e671. Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., et al. (2002). Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c. Journal of Biological Chemistry, 277, 40544e40548. Jadot, M., Colmant, C., Wattiaux-De Coninck, S., & Wattiaux, R. (1984). Intralysosomal hydrolysis of glycyl-L-phenylalanine 2-naphthylamide. Biochemistry Journal, 219, 965e970. Jha, A., Ahuja, M., Patel, S., Brailoiu, E., & Muallem, S. (2014). Convergent regulation of the lysosomal two-pore channel-2 by Mg2þ, NAADP, PI(3,5)P2 and multiple protein kinases. EMBO Journal, 33, 501e511. Kilpatrick, B. S., Eden, E. R., Schapira, A. H., Futter, C. E., & Patel, S. (2013). Direct mobilisation of lysosomal Ca2þ triggers complex Ca2þ signals. Journal of Cell Science, 126, 60e66. Lasorsa, F. M., Pinton, P., Palmieri, L., Scarcia, P., Rottensteiner, H., Rizzuto, R., et al. (2008). Peroxisomes as novel players in cell calcium homeostasis. Journal of Biological Chemistry, 283, 15300e15308. Launikonis, B. S., Zhou, J., Royer, L., Shannon, T. R., Brum, G., & Rios, E. (2006). Depletion “skraps” and dynamic buffering inside the cellular calcium store. Proceedings of the National Academy of Sciences of the United States of America, 103, 2982e2987. Lelouvier, B., & Puertollano, R. (2011). Mucolipin-3 regulates luminal calcium, acidification, and membrane fusion in the endosomal pathway. Journal of Biological Chemistry, 286, 9826e9832. Lissandron, V., Podini, P., Pizzo, P., & Pozzan, T. (2010). Unique characteristics of Ca2þ homeostasis of the trans-Golgi compartment. Proceedings of the National Academy of Sciences of the United States of America, 107, 9198e9203. Lloyd-Evans, E., Morgan, A. J., He, X., Smith, D. A., Elliot-Smith, E., Sillence, D. J., et al. (2008). Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nature Medicine, 14, 1247e1255. Lu, Y. Y., Hao, B. X., Graeff, R., Wong, C. W., Wu, W. T., & Yue, J. (2013). TPC2 signaling inhibits autophagosomal-lysosomal fusion by alkalizing lysosomal pH. Journal of Biological Chemistry, 288, 24247e24263. Lundqvist-Gustafsson, H., Gustafsson, M., & Dahlgren, C. (2000). Dynamic Ca2þ changes in neutrophil phagosomes A source for intracellular Ca2þ during phagolysosome formation? Cell Calcium, 27, 353e362. Mahapatra, N. R., Mahata, M., Hazra, P. P., McDonough, P. M., O’Connor, D. T., & Mahata, S. K. (2004). A dynamic pool of calcium in catecholamine storage vesicles. Exploration in living cells by a novel vesicle-targeted chromogranin A-aequorin chimeric photoprotein. Journal of Biological Chemistry, 279, 51107e51121. Martinez-Zaguilan, R., Parnami, J., & Martinez, G. M. (1998). Mag-Fura-2 (Furaptra) exhibits both low (microM) and high (nM) affinity for Ca2þ. Cellular Physiology and Biochemistry, 8, 158e174. McCue, H. V., Wardyn, J. D., Burgoyne, R. D., & Haynes, L. P. (2013). Generation and characterization of a lysosomally targeted, genetically encoded Ca2þ-sensor. Biochemistry Journal, 449, 449e457.

References

Mitchell, K. J., Lai, F. A., & Rutter, G. A. (2003). Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2þ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6). Journal of Biological Chemistry, 278, 11057e11064. Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., et al. (2001). Dense core secretory vesicles revealed as a dynamic Ca(2þ) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera. Journal of Cell Biology, 155, 41e51. Miyawaki, A. (2011). Development of probes for cellular functions using fluorescent proteins and fluorescence resonance energy transfer. Annual Review of Biochemistry, 80, 357e373. Mogami, H., Gardner, J., Gerasimenko, O. V., Camello, P., Petersen, O. H., & Tepikin, A. V. (1999). Calcium binding capacity of the cytosol and endoplasmic reticulum of mouse pancreatic acinar cells. Journal of Physiology, 518(Pt 2), 463e467. Moisescu, D. G., & Ashley, C. C. (1977). The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants. Biochimica et Biophysica Acta, 460, 189e205. Morgan, A. J., Davis, L. C., Wagner, K. T. Y., Lewis, A. M., Parrington, J., Churchill, G. C., et al. (2013). Bidirectional Ca2þ signaling occurs between the endoplasmic reticulum and acidic organelles. Journal of Cell Biology, 200, 789e805. Morgan, A. J., & Galione, A. (2007a). Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca2þ stores in sea urchin eggs. Journal of Biological Chemistry, 282, 37730e37737. Morgan, A. J., & Galione, A. (2007b). NAADP induces pH changes in the lumen of acidic Ca2þ stores. Biochemistry Journal, 402, 301e310. Morgan, A. J., & Galione, A. (2014). Two-pore channels (TPCs): current controversies. Bioessays, 36, 173e183. Morgan, A. J., Platt, F. M., Lloyd-Evans, E., & Galione, A. (2011). Molecular mechanisms of endolysosomal Ca2þ signalling in health and disease. Biochemistry Journal, 439, 349e374. Morgan, A. J., & Thomas, A. P. (2006). Single-cell and subcellular measurement of intracellular Ca2þ concentration. Methods in Molecular Biology, 312, 87e117. Murphy, R. F., Powers, S., & Cantor, C. R. (1984). Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. Journal of Cell Biology, 98, 1757e1762. Nguyen, T., Chin, W. C., & Verdugo, P. (1998). Role of Ca2þ/Kþ ion exchange in intracellular storage and release of Ca2þ. Nature, 395, 908e912. Palmer, A. E., Jin, C., Reed, J. C., & Tsien, R. Y. (2004). Bcl-2-mediated alterations in endoplasmic reticulum Ca2þ analyzed with an improved genetically encoded fluorescent sensor. Proceedings of the National Academy of Sciences of the United States of America, 101, 17404e17409. Palmer, A. E., & Tsien, R. Y. (2006). Measuring calcium signaling using genetically targetable fluorescent indicators. Nature Protocols, 1, 1057e1065. Pandey, V., Chuang, C. C., Lewis, A. M., Aley, P., Brailoiu, E., Dun, N., et al. (2009). Recruitment of NAADP-sensitive acidic Ca2þ stores by glutamate. Biochemistry Journal, 422, 503e512. Parkesh, R., Lewis, A., Aley, P., Arredouani, A., Rossi, S., Tavares, R., et al. (2008). Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2þ signalling in intact cells. Cell Calcium, 43, 531e538.

193

194


Penny, C. J., Kilpatrick, B. S., Han, J. M., Sneyd, J., & Patel, S. (2014). A computational model of lysosome-ER Ca2þ microdomains. Journal of Cell Science, 127, 2934e2943. Pinton, P., Pozzan, T., & Rizzuto, R. (1998). The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2þ store, with functional properties distinct from those of the endoplasmic reticulum. Embo Journal, 17, 5298e5308. Pinton, P., Rimessi, A., Romagnoli, A., Prandini, A., & Rizzuto, R. (2007). Biosensors for the detection of calcium and pH. Methods in Cell Biology, 80, 297e325. Pittman, J. K. (2011). Vacuolar Ca2þ uptake. Cell Calcium, 50, 139e146. Pouli, A. E., Karagenc, N., Wasmeier, C., Hutton, J. C., Bright, N., Arden, S., et al. (1998). A phogrin-aequorin chimaera to image free Ca2þ in the vicinity of secretory granules. Biochemistry Journal, 330(Pt 3), 1399e1404. Pryor, P. R. (2012). Analyzing lysosomes in live cells. Methods in Enzymology, 505, 145e157. Quesada, I., Chin, W. C., & Verdugo, P. (2003). Atp-independent luminal oscillations and release of Ca(2þ) and H(þ) from mast cell secretory Granules: Implications for signal transduction. Biophysical Journal, 85, 963e970. Raveh, A., Valitsky, M., Shani, L., Coorssen, J. R., Blank, P. S., Zimmerberg, J., et al. (2012). Observations of calcium dynamics in cortical secretory vesicles. Cell Calcium, 52, 217e225. Rizzuto, R., Simpson, A. W., Brini, M., & Pozzan, T. (1992). Rapid changes of mitochondrial Ca2þ revealed by specifically targeted recombinant aequorin. Nature, 358, 325e327. Robb-Gaspers, L. D., Burnett, P., Rutter, G. A., Denton, R. M., Rizzuto, R., & Thomas, A. P. (1998). Integrating cytosolic calcium signals into mitochondrial metabolic responses. Embo Journal, 17, 4987e5000. Ruas, M., Rietdorf, K., Arredouani, A., Davis, L. C., Lloyd-Evans, E., Koegel, H., et al. (2010). Purified TPC isoforms form NAADP receptors with distinct roles for Ca2þ signaling and endolysosomal trafficking. Current Biology, 20, 703e709. Sanjurjo, C. I., Tovey, S. C., Prole, D. L., & Taylor, C. W. (2012). Lysosomes shape IP3evoked Ca2þ signals by selectively sequestering Ca2þ released from the endoplasmic reticulum. Journal of Cell Science, 126, 289e300. Santodomingo, J., Vay, L., Camacho, M., Hernandez-Sanmiguel, E., Fonteriz, R. I., Lobaton, C. D., et al. (2008). Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules. European Journal of Neuroscience, 28, 1265e1274. Shen, D., Wang, X., Li, X., Zhang, X., Yao, Z., Dibble, S., et al. (2012). Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release. Nature Communications, 3, 731. Sherwood, M. W., Prior, I. A., Voronina, S. G., Barrow, S. L., Woodsmith, J. D., Gerasimenko, O. V., et al. (2007). Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells. Proceedings of the National Academy of Sciences of the United States of America, 104, 5674e5679. Simpson, A. W. (2006). Fluorescent measurement of [Ca2þ]c: basic practical considerations. Methods in Molecular Biology, 312, 3e36. Stauber, T., & Jentsch, T. J. (2013). Chloride in vesicular trafficking and function. Annual Review of Physiology, 75, 453e477. Steen, M., Kirchberger, T., & Guse, A. H. (2007). NAADP mobilizes calcium from the endoplasmic reticular Ca(2þ) store in T-lymphocytes. Journal of Biological Chemistry, 282, 18864e18871. Suzuki, J., Kanemaru, K., Ishii, K., Ohkura, M., Okubo, Y., & Iino, M. (2014). Imaging intraorganellar Ca2þ at subcellular resolution using CEPIA. Nature Communications, 5, 4153.

References

Takahashi, A., Camacho, P., Lechleiter, J. D., & Herman, B. (1999). Measurement of intracellular calcium. Physiological Reviews, 79, 1089e1125. Thomas, D., Tovey, S. C., Collins, T. J., Bootman, M. D., Berridge, M. J., & Lipp, P. (2000). A comparison of fluorescent Ca2þ indicator properties and their use in measuring elementary and global Ca2þ signals. Cell Calcium, 28, 213e223. Trollinger, D. R., Cascio, W. E., & Lemasters, J. J. (2000). Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2þ)-indicating fluorophores. Biophysical Journal, 79, 39e50. Tsien, R. Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry, 19, 2396e2404. Turk, V., Turk, B., & Turk, D. (2001). Lysosomal cysteine proteases: facts and opportunities. Embo Journal, 20, 4629e4633. Wang, X., Zhang, X., Dong, X-p., Samie, M., Li, X., Cheng, X., et al. (2012). TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes. Cell, 151, 372e383. Wu, J., Liu, L., Matsuda, T., Zhao, Y., Rebane, A., Drobizhev, M., et al. (2013). Improved orange and red Ca(2)þ/ indicators and photophysical considerations for optogenetic applications. ACS Chemical Neuroscience, 4, 963e972. Zhang, Z. H., Lu, Y. Y., & Yue, J. (2013). Two-pore channel-2 differentially modulates neural differentiation of mouse embryonic stem cells. PLoS One, 8, e66077. Zong, X., Schieder, M., Cuny, H., Fenske, S., Gruner, C., Rotzer, K., et al. (2009). The twopore channel TPCN2 mediates NAADP-dependent Ca2þ-release from lysosomal stores. Pflugers Archiv, 458, 891e899.

195

Measuring relative lysosomal volume for monitoring lysosomal storage diseases.

Lysosomal calcium regulates autophagy.

Kinetic Approaches to Measuring Peroxiredoxin Reactivity.

Approaches to measuring entanglement in chemical magnetometers.

High-throughput approaches to measuring cell death.

Measuring lysosomal pH by fluorescence microscopy.

An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.

Approaches to identifying, measuring, and aggregating elements of value.

Depression in hospitalized child psychiatry patients. Approaches to measuring depression.

The role of cell calcium in current approaches to toxicology.

Neuronopathic lysosomal storage disorders: Approaches to treat the central nervous system.

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB.

Regulation of mTORC1 by lysosomal calcium and calmodulin.

Approaches to measuring ejection fraction: Many tools, but how to decide which one?

New Approaches to Molecular Imaging of Multiple Myeloma.

Surgical approaches to central skull base and postsurgical imaging.

Approaches to imaging unfolded secretory protein stress in living cells.

Imaging Approaches to Investigate Myonuclear Positioning in Drosophila.

Percutaneous stone removal: new approaches to access and imaging.

The minimal requirements to use calcium imaging to analyze ICRAC.

Approaches for measuring signalling plasticity in the context of resistance to targeted cancer therapies.

"Which box should I check?": examining standard check box approaches to measuring race and ethnicity.

Optimising PET approaches to measuring 5-HT release in human brain.

New Approaches to Measuring Sticky Molecules: Improvement of Instrumental Response Times Using Active Passivation.