Probing Ca2 + release mechanisms using sea urchin egg homogenates

CHAPTER

Probing Ca2+ release mechanisms using sea urchin egg homogenates

19

Yu Yuana, Gihan S. Gunaratneb, Jonathan S. Marchantb,c, Sandip Patela,* a

Department of Cell and Developmental Biology, University College London, London, United Kingdom b Department of Pharmacology, University of Minnesota, Minneapolis, MN, United States c Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States *Corresponding author: e-mail address: [email protected]

Chapter outline 1 Introduction......................................................................................................446 1.1 Ca2 + release pathways........................................................................446 1.2 The sea urchin egg homogenate...........................................................446 1.3 Applications of the sea urchin egg homogenate.....................................447 1.3.1 Defining Ca2 + stores.......................................................................447 1.3.2 Defining pharmacology of Ca2 + release............................................447 1.3.3 Defining messenger binding sites.....................................................448 1.4 Advantages and limitations of the sea urchin egg preparation..................448 1.4.1 Advantages.....................................................................................448 1.4.2 Limitations......................................................................................449 2 Methods...........................................................................................................450 2.1 Preparation of egg homogenates..........................................................450 2.1.1 Materials........................................................................................450 2.1.2 Methods.........................................................................................450 2.2 Monitoring Ca2 + channel activation......................................................451 2.2.1 Materials........................................................................................451 2.2.2 Loading homogenates with Ca2 +......................................................451 2.2.3 Measuring medium [Ca2 +]..............................................................452 2.2.4 Measuring luminal Ca2 + content......................................................452 References............................................................................................................455

Methods in Cell Biology, Volume 151, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2018.10.007 © 2019 Elsevier Inc. All rights reserved.

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Abstract Sea urchin eggs have been extensively used to study Ca2+ release through intracellular Ca2+permeable channels. Their amenability to homogenization yields a robust, cell-free preparation that was central to establishing the Ca2+ mobilizing actions of cyclic ADP-ribose and NAADP. Egg homogenates have continued to provide insight into the basic properties and pharmacology of intracellular Ca2+ release channels. In this chapter, we describe methods for the preparation of egg homogenates and monitoring Ca2+ release using fluorimetry and radiotracer flux.

1 Introduction 1.1 Ca2+ release pathways Ca2+ is a key intracellular messenger regulating numerous cellular processes from the onset of life (fertilization) through to death (apoptosis) (Berridge, Bootman, & Roderick, 2003). In many cases, the all-important Ca2+ signals that drive Ca2+dependent output derive from intracellular Ca2+ stores. The ER represents a major Ca2+ store endowed with well-defined pumps to effect Ca2+ uptake, buffers to effect storage and Ca2+-permeable channels to effect Ca2+ release (Clapham, 2007). The latter include inositol trisphosphate (IP3) and ryanodine receptors which are activated by the intracellular messengers IP3 and cyclic ADP-ribose (cADPR), respectively (Berridge, 1993; Lee, 1997). In addition, a number of acidic organelles such endosomes and lysosomes also serve as Ca2+ stores. These so-called acidic Ca2+ stores are also equipped with an appropriate Ca2+ signaling toolkit, albeit one less well defined in molecular terms than its ER counterpart (Morgan, Platt, Lloyd-Evans, & Galione, 2011; Patel & Cai, 2015; Patel & Docampo, 2010). Central to acidic store Ca2+ signals are the two-pore channels (TPCs) and the Ca2+ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) (Galione, 2015; Lee, 2005; Patel, 2015).

1.2 The sea urchin egg homogenate Soon after the discovery of IP3 as a Ca2+ mobilizing messenger, Lee and colleagues demonstrated that the adenine nucleotides NAD and NADP could also mobilize stored Ca2+ (Clapper, Walseth, Dargie, & Lee, 1987). This study was performed using a homogenized preparation of sea urchin eggs bathed in an intracellular-like medium where medium Ca2+ concentration was measured using a fluorescent Ca2+ indicator. Ca2+ release persisted when the homogenates had been desensitized to IP3 pointing to alternative mechanisms of Ca2+ release. A body of subsequent work identified cADPR as a metabolite of NAD produced by ADP-ribosyl cyclases, and NAADP (a contaminant in NADP), as the active species (Galione et al., 1993; Howard et al., 1993; Lee & Aarhus, 1995; Lee, Walseth, Bratt, Hayers, & Clapper, 1989).

1 Introduction

These early studies laid the foundations for subsequent work that established these molecules as Ca2+ mobilizing messengers in cells across the natural world (Lee, 2012) coming full circle with the molecular identification of an expanded family of ADP-ribosyl cyclases in the sea urchin (Churamani et al., 2007, 2008; Davis et al., 2008; Ramakrishnan et al., 2010).

1.3 Applications of the sea urchin egg homogenate Many of the principles underpinning cADPR and NAADP action can be traced back to studies using sea urchin egg preparations (Galione, Patel, & Churchill, 2000; Lee, 1996).

1.3.1 Defining Ca2+ stores The notion that IP3 and cADPR release Ca2+ from a Ca2+ store (ER) distinct to that targeted by NAADP (acidic organelles) was first demonstrated in egg homogenates subjected to density gradient fractionation (Clapper et al., 1987). Thus, whereas IP3 and cADPR effected Ca2+ release from fractions associated with ER markers, the distribution of NAADP-sensitive fractions was much broader yielding several fractions that were uniquely sensitive to NAADP (Clapper et al., 1987). IP3 and cADPR-induced Ca2+ release from egg homogenates was blocked by thapsigargin, a Ca2+ pump inhibitor that drains Ca2+ from ER stores; NAADP-mediated Ca2+ signals however were not (Genazzani & Galione, 1996). These findings were supported by intact cell studies where cADPR and IP3 released Ca2+ from the opposite pole to NAADP in intact eggs subjected to organelle stratification by gentle centrifugation (Lee & Aarhus, 2000). Analysis of NAADP-sensitive fractions identified the reserve granules as the likely target stores (Churchill et al., 2002). It is the lysosome-like nature of these organelles that focussed subsequent efforts which culminated in the identification of the endo-lysosomal TPCs as NAADP targets. Humans and mice possess two isoforms (Brailoiu et al., 2009; Calcraft et al., 2009; Zong et al., 2009) whereas sea urchins possess three isoforms, more typical of other deuterostomes (Brailoiu et al., 2010; Cai & Patel, 2010; Ruas et al., 2010). Notably, NAADP-sensitive fractions sequestered Ca2+ in a proton-sensitive manner pointing to a putative Ca2+-H+ exchanger (Churchill et al., 2002). Recent studies have identified the genes encoding CAX proteins in the sea urchin and other animals thereby providing a molecular basis for Ca2+ uptake (Melchionda, Pittman, Mayor, & Patel, 2016).

1.3.2 Defining pharmacology of Ca2+ release The sea urchin egg preparation has played an important role in defining the basic pharmacology of cADPR and NAADP action. cADPR was shown to promote Ca2+-induced Ca2+ release in egg homogenates and to be modulated by a number of pharmacological regulators of ryanodine receptors such as caffeine, procaine and ruthenium red (Galione, Lee, & Busa, 1991; Lee, 1993). The preparation was also central to establishing the properties of 8-substituted antagonists such as 8-amino

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cADPR (Walseth & Lee, 1993) and the metabolically resistant derivative 7-deaza 8-bromo-cADPR (Sethi, Empson, Bailey, Potter, & Galione, 1997). Additional early work established that Ca2+ release by NAADP in egg homogenates was inhibited by a number of L-type voltage-gated Ca2+ channel blockers (Genazzani et al., 1997). More recent studies have shown that these blockers inhibit Ca2+ release by TPCs (Rahman et al., 2014). Docking analyses identified a putative binding site within the pore for these drugs and local anesthetics (Rahman et al., 2014). This may represent an ancestral site acquired prior to the supposed intragenic duplication of primordial TPC-like channel leading to present day four domain voltage-gated Ca2+ and Na+ channels. The widely used NAADP antagonist, Ned-19, was also first characterized using egg homogenates (Naylor et al., 2009). Here, top-ranked hits from a virtual screen of compounds with shape and electrostatic similarity to NAADP were screened for the ability to selectively block NAADP-evoked Ca2+ signals.

1.3.3 Defining messenger binding sites Binding sites for both cADPR and NAADP have been characterized in egg homogenates using 32P labeled probes (Aarhus et al., 1996; Lee, 1991). Binding of NAADP is highly unusual because NAADP appears to bind its receptor in an essentially irreversible manner in the presence of physiological levels of K+ (Aarhus et al., 1996; Dickinson & Patel, 2003). This property has been exploited to track labeled receptors, which are more stable than their unliganded receptors, during biochemical fractionation (Berridge, Dickinson, Parrington, Galione, & Patel, 2002; Churamani, Dickinson, Ziegler, & Patel, 2006). The avidity of the preparation for NAADP also forms the basis of a highly sensitive radioreceptor assay (Churamani, Carrey, Dickinson, & Patel, 2004; Lewis et al., 2007). This has allowed determination of NAADP levels in a number of cell types (Churamani et al., 2004) including sea urchin sperm (Billington, Ho, & Genazzani, 2002). Importantly, use of this assay was central to documenting changes in cellular NAADP levels during sea urchin fertilization (Churchill et al., 2003) and upon stimulation of mammalian cells with physiological extracellular Ca2+ mobilizing stimuli such as hormones, nutrients and neurotransmitters (Kinnear, Boittin, Thomas, Galione, & Evans, 2004; Pandey et al., 2009; Yamasaki et al., 2005). These findings cemented NAADP as a novel Ca2+ mobilizing messenger.

1.4 Advantages and limitations of the sea urchin egg preparation 1.4.1 Advantages Ca2+ mobilizing messengers are intrinsically cell-impermeable necessitating methods to breach the cell membrane in order to allow access to their intracellular sites of action. To prepare sea urchin egg homogenates, cells are physically disrupted in order to release organelles into the experimental medium which can then be readily supplemented with Ca2+ mobilizing messengers. Such an approach circumvents the technically demanding techniques of microinjecting intact cells or intracellular

1 Introduction

dialysis using patch pipettes. While cell-permeable analogues for all three messengers have been described (Gu et al., 2004; Li, llopis, Whitney, Zlokarnik, & Tsien, 1998; Li, Schulte, llopis, & Tsien, 1997; Parkesh et al., 2007), calibrating their intracellular concentrations is difficult and NAADP-AM is notoriously unstable. IP3-induced Ca2+ signals are readily observable in numerous cell-free preparations. But there are far fewer reports describing the actions of cADPR and NAADP in a broken cell setting. This is probably because both likely bind to accessory proteins that associate with their target channels (Lin-Moshier et al., 2012; Walseth, Aarhus, Kerr, & Lee, 1993; Walseth et al., 2012)—an association that maybe perturbed upon cell disruption. The sea urchin egg homogenate is ideal because it retains sensitivity to all three messengers. This is a major advantage as it allows probing of IP3, cADPR and NAADP action in parallel allowing the selectivity of a given manipulation (e.g., drug block) to be easily determined. Its suitability for miniaturization (e.g., 96-well format (Dickinson & Patel, 2003)) improves throughput. Another major advantage of the sea urchin homogenate is its stability. It can be prepared in bulk and stored at
80 °C for many years without appreciable loss of activity. Finally, the egg homogenate is highly reproducible. Although preparations that are insensitive to messengers can be occasionally encountered (Patel, Churchill, & Galione, 2000), a given sensitive preparation yields very similar results between labs. For example, blockade of NAADP-mediated Ca2+ signals by voltage-gated Ca2+ channel blockers published nearly two decades apart by different laboratories (Genazzani et al., 1997; Rahman et al., 2014) reported the same rank order of potency (diltiazem > verapamil > nifedipine) and similar half-maximal inhibitory concentrations (within approximately two-fold).

1.4.2 Limitations Despite these advantages, access to sea urchins is required. These animals are much less available than, for example, rodent models although once acquired they can be housed in captivity (see Adams, Heyland, Rice & Foltz, 2019). To date, most studies have been performed with wild Lytechinus pictus or Strongylocentrotus purpuratus which live off the West coast of North America/Mexico. This levies considerable shipment costs for laboratories outside of the area. And of course sea urchins are not vertebrates thus lessening their appeal for biomedical applications. While the utility of non-vertebrate models exemplified by Drosophila is considerable, differences with vertebrates are inevitable. For example, in both sea urchin and mammalian systems, NAADP-mediated Ca2+ release is subject to unusual inactivation. But in eggs, subthreshold (picolar) concentrations of NAADP inactivate Ca2+ release (Aarhus et al., 1996; Genazzani, Empson, & Galione, 1996), whereas in mammals, inactivation is observed at supra-maximal (micromolar) concentrations (Cancela, Churchill, & Galione, 1999). Pharmacological differences between NAADP analogues in releasing Ca2+ in sea urchin and mammalian cells have also been reported (Ali et al., 2014).

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2 Methods 2.1 Preparation of egg homogenates Egg homogenates can be readily prepared in bulk. In brief, the eggs are washed extensively, resuspended into an intracellular-like medium and homogenized.

2.1.1 Materials Sea urchins: L. pictus or S. purpuratus. Mesh. Nitex® 85 μm. KCl. 0.5 M in H2O. Artificial seawater (ASW). Dissolve instant ocean solid in deionized water to a final specific gravity of 1.02–1.023 using a hydrometer (approximately 36 g/L). Nominally Ca2+-free ASW. 470 mM NaCl, 27 mM MgCl2, 28 mM MgSO4, 10 mM KCl, 2.5 mM NaHCO3 (pH 8 with NaOH). Ca2+-free ASW. Nominally Ca2+-free ASW supplemented with 1 mM EGTA (pH 8). GluIM. 250 mM NMDG, 250 mM K-gluconate, 20 mM Na-HEPES, 1 mM MgCl2 (pH 7.2 with glacial acetic acid). Complete GluIM (2 ). GluIM supplemented with 2 mM MgATP, 20 mM phosphocreatine and 20 U/mL creatine phosphokinase (pH 7.2). Add EDTA-free protease inhibitor tablets to twice their normal final concentration.

2.1.2 Methods Inject KCl into each cavity surrounding the mouth of the sea urchin and shake to initiate spawning. Place urchin upside-down on the top of a tube/small beaker filled to the brim with ASW to collect eggs (orange in appearance). Discard sperm producing males. We typically process 500 L. pictus at a time. Once all urchins have been shed, decant off ASW and combine eggs. Filter through mesh into ASW to remove jelly. Centrifuge the de-jellied egg suspension at 4 °C (1000 rpm, 10 , JA-10 rotor, 100  g) and resuspend in 10 mL Ca2+-free ASW/mL packed eggs. Repeat twice to wash out Ca2+. Resuspend eggs in nominally Ca2+-free ASW and wash twice by centrifugation to ensure removal of EGTA. Resuspend eggs in GluIM which approximates the egg cytosol and wash once by centrifugation. Resuspend the eggs after the final spin in an equal volume of complete GluIM (2) to form a 50% (v/v) suspension. This solution is supplemented with an ATP-regenerating system. The following procedures are best performed in a cold room. Homogenize 15 mL aliquots of the final egg suspension with a Dounce homogenizer using a tight-fitting plunger (approximately six strokes). Aliquot into 1.5 mL tubes and pulse spin for 8 s in a microfuge to remove cortical granules. Immediately remove supernatant and pool. Prepare 500 μL, single use aliquots and store at
80 °C.

2 Methods

Ca2+

A

Fluorimetry

Messenger

B

Ca2+

45

Liquid scintillation counting

FIG. 1 Methods for monitoring Ca2+ mobilizing messenger action in sea urchin egg homogenates. Schematic of Ca2+ stores (large circles) loaded with Ca2+ (blue) either in the absence (A) or in the presence (B) of 45Ca (red). Addition of a Ca2+ mobilizing messenger (green) results in mobilization of Ca2+ through a Ca2+-permeable channel (yellow). This activity can be measured by monitoring an increase in the medium Ca2+ concentration by fluorimetry (A) or a reduction in the luminal Ca content by liquid scintillation counting (B).

2.2 Monitoring Ca2+ channel activation Egg homogenates readily take up Ca2+ from their surrounding medium in an ATPdependent manner. Addition of Ca2+ mobilizing messengers opens Ca2+ release channels which can be monitored either by measuring the ensuing increase in the medium Ca2+ concentration using a fluorescent Ca2+ indicator or the decrease in the luminal Ca2+ content using radiolabelled 45Ca (Fig. 1). Methods for both are described.

2.2.1 Materials Complete GluIM (1 ). GluIM supplemented with 1 mM MgATP, 10 mM phosphocreatine and 10 U/mL creatine phosphokinase (pH 7.2). Fluo-3 or Fluo-4 salt. 3 mM in DMSO. 45 Ca, 45 mCi/mL in H2O (specific activity 26 mCi/mg; batch specific). KCN, 100 mM in H2O. Glass fiber filters (grade GF/B). Wash buffer. 750 mM sucrose and 10 mM sodium citrate at 4 °C.

2.2.2 Loading homogenates with Ca2+ Thaw an aliquot of egg homogenate and add an equal volume of complete GluIM (1). For measuring luminal Ca2+ content supplement complete GluIM (1 ) with 0.75 μCi/mL 45Ca and 1 mM KCN (to prevent mitochondrial Ca2+ uptake) prior to

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dilution. Perform further two-fold dilutions at 30 min intervals. Ideally, these experiments are performed at 17 °C. However, we find that signals are readily measurable at room temperature. For fluorometric determinations, supplement the homogenate with Fluo-3/Fluo-4 to a final concentration of 3 μM at the last dilution step. We typically use homogenates at a final concentration of 1.25–2.5% (v/v). In our hands, homogenates derived from S. purpuratus produce robust Ca2+ responses when used at 1.25% (v/v). However, homogenates derived from L. pictus may require concentrations up to 5% (v/v). For 45Ca, serially dilute the homogenate with complete GluIM (1 )/45Ca2+ to a final concentration of 0.625% (v/v). We find that step-wise dilutions improve subsequent sensitivity to Ca2+ mobilizing messengers. Alternative dilution protocols, for example, an initial 1:5 dilution (1 h), followed by 1:2 dilutions (1 h each) also produce highly sensitive preparations.

2.2.3 Measuring medium [Ca2+] Medium Ca2+ concentration can be readily measured in real time by monitoring the fluorescence of a Ca2+ indicator in a standard fluorimeter or a fluorescence plate reader. We typically use the non-ratiometric indicators Fluo-3 or Fluo-4 (excitation/emission wavelengths of 506/526 nm and 494/506 nm, respectively). Any other fluorescent Ca2+ indicator can be used as long as it is in the cell impermeant form (and not the more common ester which is only suitable for intact cell studies). For cuvette-based fluorimetry, we use small volume cuvettes to conserve on homogenate typically analyzing 600 μL of Ca2+-loaded homogenate in a Perkin Elmer LS50B Fluorimeter. Add Ca2+ mobilizing messengers from stock solutions at 100 their final concentration (in H2O) and mix by pipetting using a P200 Gilson pipette. For plate reader-based fluorimetry, we use clear-bottom, black-walled 96-well plates (to reduce fluorescence crosstalk between wells) in a Tecan Infinite M1000 Pro plate reader. With this instrumentation, recordings can be made using 200 μL of homogenate. Add Ca2+ mobilizing messengers from 40  stocks (prepared in GluIM) using a multi-channel pipette and mix using the shake function. Display time-courses in arbitrary fluorescence units (F) or normalize data to basal fluorescence values acquired prior to stimulation (F/F0). Fig. 2 shows exemplar data obtained by cuvette-based fluorimetry (Fig. 2A) or a plate reader (Fig. 2B-F). NAADP, cADPR and IP3 all evoke robust release in a concentration-dependent manner.

2.2.4 Measuring luminal Ca2+ content Luminal Ca2+ content can be monitored by measuring the accumulation of 45Ca into vesicular fractions. This is achieved by loading homogenates with Ca2+ in the presence of 45Ca and separating the particulate fraction (containing sequestered 45Ca) from the medium (containing the majority of the radiolabel) by rapid filtration. Sequestered 45Ca2+ is measured by liquid scintillation counting. We use a 24 sample vacuum filtration system (Brandel). Prior to filtration, purge the vacuum filtration system with ice cold wash buffer. Place Ca2+ mobilizing

2 Methods

A

fluo-4 F/F0

6

IP3

GlulM ctrl

3

0

200 Time (s)

400

C

D

[NAADP], nM

3000

GlulM Ctrl 10 30 100 300 1000 3000 10,000

[cADPR], nM

6000

fluo-3 RFU

6000

GlulM Ctrl 10 30 100 300 1000 3000 10,000

3000

0

0 0

400 Time (s)

800

E

0

400 Time (s)

800

F

6000 [IP3], nM GlulM Ctrl 10 30 100 300 1000 3000 10,000

3000

4

NAADP cADPR IP3

Peak F/F0

fluo-3 RFU

cADPR

[Agonist]

0

fluo-3 RFU

NAADP

B

1 mM NAADP

2

0

0

400 Time (s)

800

10–9

10–8 10–7 10–6 [Agonist], M

10–5

FIG. 2 Measuring medium [Ca2+] in response to Ca2+ mobilizing messengers in sea urchin egg homogenates. (A) Typical response of a Lytechinus pictus egg homogenate to NAADP (1 μM) monitored with Fluo-4 by cuvette-based fluorimetry. Data are presented as F/F0 values. (B) Typical responses of a Strongylocentrotus purpuratus egg homogenate to increasing concentrations of NAADP, cADPR and IP3 monitored with Fluo-3 in a 96-well plate. (C-E) Averaged traces evoked by the indicated Ca2+ mobilizing messenger at the indicated concentration presented as raw fluorescence values. (E) Concentration-effect relationships, plotted using peak F/F0 values from each experimental condition in (C-E).

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messenger from a 100  stock solution (in H2O) in 4 mL tubes. Add samples of the homogenate (typically 200 μL) to initiate mixing. Filter samples after 2 min by which time Ca2+ release is usually complete. Wash rapidly three times with wash buffer (2 mL/wash) to ensure complete separation of sequestered and free 45Ca. Gently collect each filter using tweezers and transfer to a vial containing 5 mL of scintillation fluid. Incubate for at least 1 h. Determine the radioactivity retained on each filter using a Beckman LS6000SC scintillation counter. Present data as CPMs or calibrate to absolute 45Ca levels taking into account the specific activity. Fig. 3A shows exemplar data using this method. NAADP (1 μM) typically reduces luminal 45Ca content by 30% relative to ionomycin (10 μM), a Ca2+ ionophore. This reduction is prevented by the voltage-gated Ca2+ channel blocker nifedipine in a concentration-dependent manner (Fig. 3B).

DMSO

Nifedipine

Ionomycin

30000

20000

10000

45

Ca content (CPM)

A

0

–

+

– NAADP

+

–

B NAADP response (%)

454

100

50

0 0.1

10

1000

Nifedipine (mM)

FIG. 3 Measuring luminal Ca2+ content in response to Ca2+ mobilizing messengers in sea urchin egg homogenates. (A) Typical 45Ca content measurements of Lytechinus pictus egg homogenate stimulated with 1 μM NAADP or 10 μM ionomycin in the presence of DMSO or 90 μM Nifedipine. (B) Concentration-effect relationship for nifedipine. Data are normalized to the reduction of the 45Ca content by NAADP in the presence of DMSO.

References

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