Predicting and avoiding subcellular ...

Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart K Thompson1, P Dockery1, RW Horobin2 of Anatomy, School of Medicine, NUI Galway, Ireland, and 2School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK

Biotech Histochem Downloaded from informahealthcare.com by University of Glasgow on 10/02/12 For personal use only.

1Discipline

Accepted June 13, 2012

Abstract Stimulated by difficulties experienced when using fluo-3 AM, we developed a general mechanistic model to aid understanding and practical application of calcium probes applied as acetoxymethyl (AM) esters. Several practical issues previously overlooked or under-emphasized are considered by this model. First, some AM ester probes are “super” lipophilic, e.g., calcium orange, fluo-3, fura red, and these are trapped in the plasma membrane. Entry of such compounds into cells requires the presence of serum albumin in the incubation medium or esterase in the plasma membrane or both. Second, visible cytosolic calcium signals require significant cytosolic esterase, which varies considerably among cell lines and within cell populations of a single cell line. Finally, compartmentalization artefacts are most likely when incompletely hydrolyzed esters are present in the cytosol. This can occur because of low cytosolic esterase concentration or activity, and especially when long incubation times or high extracellular probe concentrations are used. An additional factor favoring compartmentalization is the presence of the “salt” form of the probe in the cytosol in the absence of significant concentrations of calcium ions. We provide an algorithmic chart to aid assessment of possible compartmentalization, guides to relevant QSAR models, and notes on estimation of the structural parameters required when using these models. Key words: calcein, calcium green-1, calcium orange, fluo-3, fluorescent probe, fura-2, fura red, indo-1, QSAR model, quin-2, rhod-2 Fluorescent probes for cytosolic calcium are applied widely in current biology as indicated by several reviews of the methodology and its applications (Adams 2010, Homma et al. 2009, Paredes et al. 2008, Swanson et al. 2011, Thomas et al. 2000). Because the calcium chelating agents in such methods are polycarboxylated and hydrophilic, they do not readily penetrate cell membranes. A routine tactic for circumventing this problem is to Correspondence: Dr. K. Thompson, Discipline of Anatomy, School of Medicine, NUI Galway, Galway, Ireland. E-mail: kerry. [email protected] © 2012 The Biological Stain Commission Biotechnic & Histochemistry 2012, 87(7): 468–483.

DOI:10.3109/10520295.2012.703691

apply the probes as carboxylic acid esters, typically acetoxymethyl (AM) esters. These compounds generally are considered membrane permeable and their conversion into the active polycarboxylated species is achieved by cytosolic esterases. This simple model, illustrated diagrammatically in Fig. 1, makes various assumptions, e.g., that only completely esterified and completely deesterified probe species need be considered, and that cytosolic de-esterification is complete and rapid, which results in trapping of membrane impermeable, completely de-esterified compounds in this cellular region. These assumptions sometimes are valid and such procedures often are technically non-problematic. 468


Fig. 1. Mechanism of action of AM ester calcium probes described by the simple model. Chemical structures are illustrative only.

Complications and artefacts have been described, however, for all the steps in the process. Although not always made explicit in accounts

of the simple model, the following scenarios have been reported. 1) The extracellular probe may be incompletely esterified due to either the

Predicting compartmentalization artefacts of calcium probes 469


presence of impurities in purchased reagents (Kawanishi et al. 1989) or the occurrence of ester hydrolysis in the incubation media (Persidsky and Baillie 1977, Jobsis et al. 2007). 2) Not all AM esters are membrane permeant; some are super lipophilic and could accumulate in the plasma membrane (Wright et al. 1996). 3) Even a completely de-esterified probe could enter cells by pinocytosis (Dustin 2000, Tenopoulou et al. 2007). 4) Wherever ester hydrolysis takes place, it occurs in a stepwise manner, which gives rise to a series of compounds with variable numbers of ester and carboxylic acid moieties. For example, hydrolysis of fluo-3 AM could yield 16 distinct products, many of which are structural isomers, and each carboxylic acid group can exist either as an anion or a free acid. Consequently, the components of such a series vary markedly in electric charge, lipophilicity and amphiphilicity as shown schematically in Fig. 2 and indicated numerically in Table 1. 5) In some cell lines, cytosolic esterase concentrations may be low and significant amounts of AM ester, or of partially de-esterified compounds, can be present within the cells (Leroy et al. 1996, Oakes et al. 1988). 6) Compartmentalization of calcium probes in a variety of organelles has been reported, e.g., in endoplasmic reticulum (Williams et al. 1985), endosomes/lysosomes (Di Virgilio et al. 1990), mitochondria (Roe et al. 1990), nuclei (Malgaroli

et al. 1987), and secretion granules (Almers and Neher 1985). From the viewpoint of the laboratory scientist wanting to use fluorescent calcium probes, certain take-home messages are especially important. First, not all AM esters of calcium probes are membrane permeant. Second, multiple calcium probe species may be present under some circumstances, both extracellularly and in the cytosol. Third, under certain staining conditions, calcium probes can accumulate in a variety of cell organelles. Despite published reports like those cited above, and indeed reviews discussing such artefacts (Hoyland 1993, Moore et al. 1990), not all bench workers are aware of the potential problems. Moreover, the research reports and reviews cited here do not provide a systematic treatment of compartmentalization into non-cytosolic organelles in which, for example, the consequences of extracellular de-esterification, trapping of AM esters in the plasma membrane, and low esterase and cytosolic calcium ion concentrations are acknowledged. Consequently, after our own experience of experimental difficulties with the calcium probe fluo-3 AM, we sought to establish a systematic scheme to predict for any given calcium probe which organelles might give rise to artifactual staining of calcium. Ideally, such a scheme would not only spell out the compartmentalization patterns

Fig. 2. Illustration of potential species arising from an AM ester calcium probe owing to progressive hydrolysis and variation in pH. The diagram is generic; no particular probe is implied. In actuality the number of possible species in actual probes is much greater than shown, because AM esters possess at least twice as many acetoxymethyl moieties as illustrated.

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Biotechnic & Histochemistry 2012, 87(7): 468–483

Table 1. Key physicochemical properties and predicted intracellular localizations of fully, partially and totally de-esterified species of various widely used calcium probes


Probe and

species1

Calcein Esterified Part de-esterified Least anionic Most anionic De-esterified salt Calcium green-1 Esterified Part de-esterified Least anionic Most anionic De-esterified salt Calcium orange Esterified Part de-esterified Least anionic Most anionic De-esterified salt Fluo-3 Esterified Part de-esterified Least anionic Most anionic De-esterified salt Fura-2 Esterified Part de-esterified Least anionic Most anionic De-esterified Fura red Esterified Part de-esterified Least anionic Most anionic De-esterified salt Indo-1 Esterified Part de-esterified Least anionic Most anionic De-esterified salt Quin-2 Esterified Part de-esterified Least anionic Most anionic De-esterified salt

Z2 0 1 2 6 0 1 3 6 0

Log

P3

5.4 3.1 1.0 12.0

2.3 2.5 8.7

0

11.2

1 4 5

9.1 0.3 4.3

5.6 3.2 9.0

0

10.3

0 3 4

5.5 1.6 4.4

0 3 5 0 0 3 4

} } }

Lysosomes; mitochondria Nuclei (histones)

One of several possible ester hydrolysis products

Impermeant; plasma membrane

A zwitterion


Possible hydrolysis product; some such may go to ER (see note 6)


Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)

Permeant

}




}




}

6.4 4.8 2.3 5.2

Possible hydrolysis product; some may go to ER (see note 6)


8.7 7.2 1.6 7.4

Lysosomes; mitochondria Nuclei (histones) Permeant; biomembranes

7.0

0 3 5

0

}

9.1 7.5 0.4 2.5

Comments

Permeant; biomembranes

7.0

0 1 4

0

Predicted membrane permeance4 and accumulation sites5



Permeant; biomembranes

}


Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6) (Continued)


Table 1. (Continued) Probe and species1 Rhod-2 Esterified Part de-esterified Least anionic Most anionic De-esterified salt

Z2

Log P3

1

5.8

1 2 3

4.2 2.2 0.7

Predicted membrane permeance4 and accumulation sites5

Comments

Permeant; mitochondria Mitochondria

One of several possible AM ester hydrolysis products Hydrolysis products can have both positive and negative charges


1Esterified

indicates that all carboxylic acids are present in esterified form; part de-esterified indicates that at least one ester grouping was hydrolyzed; de-esterified salt indicates total conversion of all esters to carboxylic acids. 2Z is the maximum possible electric charge for the species. 3Log P is the logarithm of the octanol-water partition coefficient. 4Membrane permeability predicted using QSAR models summarized in Table 2. 5Sites of accumulation predicted using QSAR models summarized in Table 3. 6The hydrolysis products of all listed probes except rhod-2 include several compounds that are lipophilic weak acids, hence could accumulate in lysosomes and mitochondria. Moreover, the hydrolysis products of all probes except calcium green-1 and rhod-2 include lipophilic and moderately amphiphilic compounds that might accumulate in the endoplasmic reticulum; because the presence of such a species cannot be assumed, however, accumulation in endoplasmic reticulum cannot be predicted reliably.

expected for existing probes, but also would predict the expected localizations of any novel smallmolecule calcium probes yet to be synthesized. We conducted co-localization studies using organelle-specific probes to investigate the compartmentalization we observed with fluo-3 AM. To devise a systematic and generic model for calcium probes, we used quantitative structure activity relations (QSAR) models to relate the chemical structures of the various probe species with both their intracellular localization and their membrane permeability. For general accounts of such models, see Horobin (2001, 2010) and for decision logic tables relating physicochemical properties (modeled by structural parameters) with membrane permeability and with uptake into a variety of cell organelles, see Tables 2 and 3, respectively.

Material and methods Cultured cells We used KLE endometrial epithelial adenocarcinoma cells (ATCC Product No. CRL-1622). Culture media Dulbecco’s modified Eagle’s medium (DMEM)/ nutrient mixture F-12 Hams, phenol free (Sigma, Wicklow, Ireland; product no. D2906-1L), fetal bovine serum (GIBCO, Life Technologies, Grand Island, NY; product no. 10270-106), L-glutamine (Sigma; 472

product no. G5763), penicillin (Sigma; product no. P3032), sodium bicarbonate (Sigma; product no. S5761-500g), streptomycin (Sigma; product no. S9137), MatTec® glass bottomed dishes (Ashland, MA; product no. P35-1.0-14-C). Staining solutions Fluo-3 AM (Sigma; product no. 46394), concentrated 10.1 M hydrochloric acid (Fischer, Dublin, Ireland; product no. H/1100/PB17), Mayer ’s hemalum (BDH, VWR, Leicestershire, UK; product no. 350604T), α-naphthyl acetate (Sigma; product no. 916), neutral red (Merck, Darmstadt, Germany; C.I. 50040), rhodamine 123 (Invitrogen, Dublin, Ireland; product no. R302), fuchsin (Merck; C.I. 42510), sodium nitrite (Sigma; product no. 490814).

Cell culture KLE cells were grown in DMEM/F-12 Hams phenol free medium supplemented with 1 ml of penicillin (100 U/ml, 0.027 M)/streptomycin (100 μg/ml, 0.025 M), 1 ml L-glutamine (0.2 M) and 10% fetal calf serum. The cells were grown in T-25 or T-75 polystyrene culture flasks containing 5–10 ml of culture medium. Cells were equilibrated with 5% CO2-95% O2 and kept at 37° C. The medium was renewed three times weekly. Cells were seeded at a concentration of 2 105 cells/ml and used when they reached 60–80% confluence. For fluo-3 AM alone and for


Table 2. Decision logic table based on structured parameters and published QSAR models for predicting membrane permeability and binding to plasma membrane of calcium probes If

And if

And if

log P 8 CBN 40


AI 8 8 log P 0

CBN 40

log P 0

CBN 40

AI 8

Then Impermeable; trapped in plasma membrane Impermeable; trapped in plasma membrane Impermeable; trapped in plasma membrane Permeable; enters cytosol Impermeable; excluded

co-localization experiments, cells were imaged in MatTec® glass bottom dishes. Staining Fluo-3 AM staining Fluo-3 AM was used at a concentration of 2 μl/ml in the loading solution. Cells were stained by

aspirating the culture medium and rinsing with 2 ml of pre-warmed buffer. Cells then were incubated with fluo-3 AM solution for 25 min at 25° C in darkness, because fluo-3 AM is photosensitive. The dye solution was aspirated and the cells were rinsed twice with buffer (n 4 dishes). Co-localization with neutral red and rhodamine 123 For neutral red staining, 1 mg/ml neutral red stock solution was stored in the dark at 4° C and used at a dilution of 1:2 104. Dye solution was added to the cells and incubation was continued for 15 min at 37° C. Cells were rinsed three times with buffer, which was replaced with 2 ml of fresh buffer for observation (n 3 dishes). For Rhodamine 123 staining, rhodamine 123 was prepared as a 1 mg/ml stock solution and used at a dilution of 1:100. Staining was carried out as in the protocol above with rhodamine 123 substituted for neutral red (n 3 dishes). Combined fluo-3 AM and neutral red/ rhodamine 123 staining was carried out by first loading cells with fluo-3 AM, then staining with neutral red or rhodamine 123 for 15 min at 25° C as above. Cells were rinsed and fresh buffer was applied before imaging (n 3 dishes for each combination).

Table 3. Decision logic table based on numerical structure parameters and published QSAR models for predicting compartmentalization of calcium probes in cell organelles. For models and parameters, see Horobin (2010) If

And if

And if

Z0 6 log P 0

5 log P 0 6 AI 3.5

8 log P 5 Z0

Or if 8 AI 5 0 log Pcation 5

10 pKa 6

Zmost ionized 0

log Pfree acid 0

pKa 7 3

Log P 0

CBN 40

Z0

5 log P 0

pKa 12

Zmost ionized 0

5 log Pleast ionized 0

pKa 7 3

Zmost ionized 0

CBN 40

log P 10

40 CBN 16

8 log Panion 0

Or if Z0

CBN 40

And if

Then probe will accumulate in [mechanistic thumbnail]

AI 3.5

Cytosol [residual location] Endoplasmic reticulum [fluid membrane partitioning] Generic biomembranes [partitioning] Lysosomes [weak base ion-trapping] [precipitation trapping]

Or if CBN 40 & log P 10

[uptake by pinocytosis]

Mitochondria [electrical potential and/or cardiolipin complexation] Mitochondria [weak acid ion-trapping] Nucleus [histone binding]


Nonspecific esterase staining 2 105

Cells were seeded at a concentration of cells/ml on 13 mm round coverslips in a 24-well plate and cultured until 60–80% confluence (n 5). Solution a


Fuchsin (1 g) was dissolved in distilled 20 ml water and 5 ml of concentrated (10.1 M) hydrochloric acid was added. This solution was stirred and heated gently. After cooling, the solution was filtered and stored as aliquots at 4° C. Solution b Incubating solution 1 was prepared by adding 0.4 ml of solution a (fuchsin) drop wise, shaking well after each addition, to 0.4 ml of sodium nitrite solution (40 mg/ml) until the solution was corn colored after standing for at least 1 min. Incubating solution 2 was prepared by combining 0.25 ml of α-naphthyl acetate solution (10 mg/ml in acetone) with 7.25 ml 0.2 M phosphate buffer (2.83 g Na2HPO4/100 ml of distilled water) and 2.5 ml of distilled water. Incubating solution 1 was added to incubating solution 2; the resulting solution was filtered and used immediately. Two hundred microliters of dye solution was added to buffer-rinsed cells and incubated for 10 min at 37° C. Cells were rinsed, then fixed with 4% w/v paraformaldehyde in phosphate buffered saline. Counterstaining was with filtered Mayer ’s hemalum for 5–10 min followed by rinsing with buffer. Cells were dehydrated through graded alcohols and cleared with xylene. The coverslips were mounted on slides using DPX and air dried overnight before inspection. This protocol was adapted from Gomori (1950) and Davis and Ornstein (1959).

488 and 561 nm lasers, which produced green and red fluorescence. For analysis, we used MacBiophotonics Image J™ software (Hamilton, ON). Images were imported, corrected for background fluorescence and separated into their respective channels, i.e., images gathered using the 488 nm laser for fluo-3 staining and images gathered using the 561 nm laser showed the neutral red lysosomal and rhodamine 123 mitochondrial components. Cells also were imaged using differential interference contrast microscopy. Esterase staining Images were captured at magnifications of 10, 20 and 40 x using an inverted Olympus BX61 microscope in a bright field configuration with an Olympus DP70 camera (Mason Technology, Dublin, Ireland). Using QSAR models to predict cell localizations of calcium probes Estimation of structure parameters Lipophilicity/hydrophilicity was modeled by the log P value, i.e., the logarithm of the octanolwater partition coefficient, and amphiphilicity by the AI value (amphiphilicity index). These parameters were estimated as described by Hansch and Leo (1979) and Christensen et al. (1999), respectively. Other parameters including the conjugated bond number (CBN) and electric charge (Z) were estimated as described by Horobin (2001). Prediction of dye uptake and intracellular localization

Microscopy

Prediction of dye uptake and intracellular localization was achieved by inserting the parameters listed in Table 1 into the various decisionrule QSAR models shown in Tables 2 and 3.

Fluo-3 AM staining and co-localization experiments

Results

Images were captured on an Olympus Spinning Disk Microscope (Andor, Belfast, Northern Ireland) using 40 x oil (NA 1.3) and 60 x oil (NA 1.42) immersion objective lenses and Andor IQ software (Belfast, Northern Ireland). Prior to collection, exposure time and EM gain were set and recorded to the channel of illumination. The live cell imaging chamber was adjusted to 37° C in a 5% CO2 buffered environment. Illumination was by

474

Fluo-3 AM staining patterns As illustrated in Fig. 3, KLE cells were remarkably variable in their appearance after loading with fluo-3 AM. More than two thirds of the cells showed little or no staining. Those that did stain were either weakly labeled, with “speckled” cytoplasms, or when intensely stained, both cytoplasmic granules and nuclei typically were prominent as was



Fig. 3. Compartmentalization artefacts seen when staining KLE cells with fluo-3 AM. 60 x Objective. a) Imaging using confocal microscope fluorescence only. b) Superimposed fluorescence and differential interference contrast images. Scale bars 50 μm.

background cytosolic staining. These outcomes were not influenced by the temperature of dye loading. Co-localization staining Neutral red Some cytoplasmic granules were stained only with the lysosomal marker neutral red, some only with fluo-3 AM, and some accumulated both dyes as illustrated in Fig. 4b. Specimens bleached very quickly during image acquisition. Rhodamine 123 Some cytoplasmic granules were stained only by the mitochondrial marker, rhodamine 123, some only with fluo-3, and some accumulated both dyes as shown in Fig. 4d. Esterase staining Cellular regions containing nonspecific esterases were stained red/brown; nuclei and regions of cytoplasmic ribosomes were counterstained blue/purple. The esterase staining showed considerable cell to cell variation; more than two thirds of the cells showed little or no staining. Esterase appeared in the perinuclear regions of the cells and dividing cells displaying the most intense staining (Fig. 5). Cells exposed to the no-substrate control, however, did not contain red/brown nonspecific esterase staining (Fig. 5b).

Structure parameters and predicted cell localizations The estimated values of the structure parameters for Z, CBN and log P are listed in Table 1 for various calcium probes. The intracellular localizations predicted by the QSAR models for various probe species also are listed in Table 1.

Discussion Fluo-3 AM staining artefacts–a typical (?) example As illustrated in Fig. 3, we observed two types of staining that we regarded as artefacts. There were positive artefacts in which fluorescence due to calcium did not usually occur as generalized cytosolic staining, but instead appeared as cytoplasmic granule staining and staining of cell nuclei. There were also negative artefacts in which more than two thirds of the cells in the monolayer showed no staining of calcium. The site of the granular staining was investigated by co-localization experiments using fluorescent probes for both lysosomes and mitochondria. The staining outcomes were complex. Fluo-3 accumulated in some lysosomes and mitochondria, but other organelles of these types did not contain the probe. The possibility that the failure of many cells to stain was due to a lack of cytosolic esterase was investigated by staining a monolayer of cells using an enzyme histochemical method to demonstrate nonspecific esterase. It was apparent



Fig. 4. Co-localization staining of KLE cells with fluo-3 and two organelle probes. a) Staining with the lysosomal probe, neutral red, only. b) Sequential staining with fluo-3 AM followed by neutral red. Yellow-orange areas indicate the presence of both dyes. c) Staining with the mitochondrial probe rhodamine 123 only. d) Sequential staining with fluo-3 AM followed by rhodamine 123. Yellow-orange areas indicate the presence of both dyes. Confocal microscope fluorescence images, 40 x oil objective. Scale bars 10 μm.

that most cells did not stain strongly for esterase. Intriguingly, the proportion of cells that stained strongly for esterase was similar to the proportion of cells that stained strongly for calcium (Figs. 3 and 5). This suggests that the cells contained widely different amounts of nonspecific esterase and in the absence of this enzyme significant staining of calcium did not occur. These observations are inconsistent with the simple model. In particular, within the cells that showed calcium signals, the calcium probe had been compartmentalized into lysosomes, mitochondria and nuclei. This compartmentalization paralleled numerous earlier reports, some concerning fluo-3, but also concerning other calcium probes as illustrated by the cases cited in Table 4. Consequently, the variety of compartmentalization seen with fluo-3 AM may be regarded as typical for a calcium probe. Based on these observations, 476

we attempted to describe a general mechanism for esterified calcium probes and their interactions with cells to rationalize these observations and to permit prediction of which dyes might be compartmentalized within which organelles.

A general calcium probe staining model in which the “simple model” is a subset The fact that calcium probes are widely and often successfully used suggests that the account of calcium staining provided by the simple model often is correct. Consequently, a more general viewpoint that accounts both for the usual and successful applications and for the reported complications and artefacts must include the simple model as a special case. We outline below such an inclusive general model.



Fig. 5. Demonstration of nonspecific esterase in KLE cells using an azo dye enzyme histochemical procedure. a) Positive staining with presence of esterase indicated by a purple-red coloration. b) A no-substrate control with an absence of enzyme staining. In both cases, nuclei were counterstained using Mayer’s hemalum. 40 x objective. Scale bars 50 μm.

When a solution of an AM ester is applied to live cells, what probe species are present extracellularly? Consider in particular the consequences of incubation media that contain serum albumin (SA). In addition to any beneficial effects on living cells, “inclusion of … serum albumin … improves the solubility of AM esters in aqueous media” (Dustin 2000). Although it is known that SA allows solubilization of hydrophobic compounds owing to binding to hydrophobic regions of the macromolecule (Alvarez-Pedraglio et al. 2001), an additional solubilization mechanism must be considered in the cases of SA. SA possesses esterase activity. Consequently, probe solutions made up in media containing SA may contain deesterified, hence more hydrophilic, compounds (Persidsky and Baillie 1977, Jobsis et al. 2007). Which probe species are membrane permeant? In particular, are all fully esterified AM esters membrane permeant? As suggested earlier (Wright et al. 1996), they probably are

not. Table 1 suggests that the AM ester forms of a number of widely applied calcium probes are super lipophilic with log P 8. The QSAR models relating membrane permeability to chemical structure (Table 2; Horobin 2010, Fig. 11.1) predict that calcium orange AM, fluo-3 AM, fura red AM and indo-1 AM are trapped within the first cell membrane they contact, i.e., the plasma membrane, hence they do not enter the cytosol. At which cellular sites can de-esterification take place? Published reports (e.g., Böcking and von Deimling 1976, Dvorak et al. 1987, Monahan 1981, Monahan-Early et al. 1987) suggest that nonspecific esterases commonly occur in the cytosol, endoplasmic reticulum and the contiguous nuclear envelope, endosomes/lysosomes, mitochondria, and the plasma membrane. Although nuclear localization also has been claimed, “many authors consider nuclear esterase activity to be due to contamination by large fragments of endoplasmic reticulum” or “a specific enzyme in the nuclear envelope” (Durrer et al. 1991). How might de-esterification, however and wherever this occurs, influence membrane permeability? Because de-esterification can occur extracellularly, in the plasma membrane, in the cytosol, or in cell organelles, we shall deal with these locations separately. Extracellular de-esterification can occur, as noted above, owing to incubation media containing SA. De-esterification, therefore, can be influenced by the presence or absence of SA, by variables including time and temperature of incubation, and by the amount of time a working dye solution has stood at room temperature prior to use. Consider some examples. If an AM ester is super lipophilic, and therefore membrane impermeant, then de-esterification may generate fewer lipophilic species and increase permeability. For example, fluo-3 AM (log P 11.2) is impermeant, while partially de-esterified products that have lost several ester groups and have log P values in the range 0-5 diffuse passively across cell membranes (Table 2). If de-esterification proceeds further, however, in the extreme to the fully de-esterified salt, then the resulting hydrophilic species is membrane impermeant (Table 2). The fluo-3 pentacarboxylate salt, for example, has a log P value of -4.3. Even salts of this type, however, can enter a cell by pinocytosis, as has been reported for calcein (Tenopoulou et al. 2007). De-esterification within the plasma membrane may occur in some cell lines. Because plasma


Table 4. Examples of compartmentalization following application of the AM ester forms of some commonly used calcium probes Organelle in which dye accumulated

Dye Calcein AM Calcium green-1 AM Calcium orange AM


Fluo-3 AM

Fura-2 AM

Fura red AM

Indo-1 AM

Quin-2 AM Rhod-2 AM

Endosome/lysosome Mitochondrion Mitochondrion Nucleus Endoplasmic reticulum Mitochondrion Endoplasmic reticulum Endo-/lysosome Mitochondrion Nucleus Endoplasmic reticulum Endosome/lysosome Mitochondrion Nucleus Endoplasmic reticulum Mitochondrion Nucleus Mitochondrion Microsome (endoplasmic reticulum?) Nucleus Endosome/lysosome Mitochondrion

Example references Crowe et al. (1995) Collins et al. (2002), Dedkova and Blatter (2005) Babcock et al. (1997), Thomas et al. (2000) Babcock et al. (1997), Thomas et al. (2000) Thomas et al. (2000) Thomas et al. (2000) Thomas et al. (2000) Kuba et al. (1991), Nathanson and Burgstahlen (1992), Thomas et al. (2000) Thomas et al. (2000) Peng et al. (2009), Thomas et al. (2000) Williams et al. (1985) Di Virgilio et al. (1990), Malgaroli et al. (1987), Roe et al. (1990) Davis et al. (1987), Roe et al. (1990), Roe et al. (1990), Malgaroli et al. (1987), Williams et al. (1985) Thomas et al. (2000) Thomas et al. (2000) Broder et al. (2007), Thomas et al. (2000) Lee et al. (1988), Spurgeon et al. (1990) Lee et al. (1988), Wahl et al. (1990) Lee et al. (1988), Wahl et al. (1990) Goligorsky et al. (1986) Alano et al. (2002), Babcock et al. (1997), Nutt et al. (2002)

membrane-localized nonspecific esterase is located in the outer leaflet, cell penetration may be facilitated by transformation of AM esters taken into the membrane into less lipophilic species, which then diffuse through the membrane into the cytosol. De-esterification within the cytosol is well known and is, of course, assumed in the simple model. What is less often discussed is that cell lines show dramatic variation in cytosolic esterase activity (Lund-Pero et al. 1994), e.g., “Liver gave a 10-fold higher … activity compared with colonic cytosol which in turn showed a degree of activity approximately 10-fold higher than the barely detectable activity in brain cytosol. Breast and stomach cytosols gave mean values about half that of the colonic.” These results referred to human tissues, but were found to parallel findings in laboratory mammals. Taking the above into account, compartmentalization of calcium probes could arise due to conversion of unhydrolyzed or incompletely hydrolyzed, membrane permeant ester species to the membrane impermeable, fully hydrolyzed salts, which become trapped within the membrane-bound organelles that contain esterases. This explanation may be relevant for compartmentalization within 478

the endoplasmic reticulum, endosomes/lysosomes, and mitochondria. Given the probable absence of nuclear nonspecific esterase, however, nuclear compartmentalization must have some other mechanism. The QSAR models available for predicting cell sites for dye accumulation based on the physicochemical character of the dye can be applied to this question. As seen in Table 3, where the QSAR models are expressed in terms of decision tables, nuclear localization is predicted to occur for hydrophilic anions with a moderate degree of conjugation owing to binding to the basic histone proteins (for information on the models, see Horobin 2001, 2010, Fig. 11.2). Indeed, given the variety of species that sometimes may be present in the cytosol, compartmentalization may be enhanced by the organelle-selective accumulation of various probe species in various organelles; for details, see Table 3, and Horobin (2001, 2010, Fig. 11.2). The predictions for the various species of some widely applied calcium probes are given in Table 1. One compartment not discussed above is the endoplasmic reticulum. The QSAR decision rule model for accumulation in this organelle



suggests that the key requirements are probes that are moderately lipophilic and amphiphilic (Table 3). Although various partly de-esterified species of several probes could possess such features, firm predictions cannot be made, because the asymmetry underlying amphiphilicity arises only according to particular patterns of de-esterification and no experimental evidence is available to assess whether, in fact, this occurs. Unfortunately, compartmentalization in endoplasmic reticulum cannot be addressed by the current general model. Pulling these points together, it is possible to envisage a general inclusive model of what may occur when calcium probes interact with live cells. A slightly simplified graphic summary of such a model is given in Fig. 6. Compare Figs. 1 and 6 to see how the simple model has been enlarged, but not negated. Are all the divergences from the simple model to be regarded as problematic? Ester hydrolysis can occur prior to a probe’s reaching the cytosol, due to either the esterase activity of SA in the incubation medium or the presence of nonspecific esterase in the outer leaflet of the plasma membrane. If a membrane permeant probe is partially de-esterified to yield other permeable species, no marked changes in behavior are expected. If a super lipophilic probe, e.g., fluo-3 AM, is converted to a less lipophilic, more permeable species, then probe loading is facilitated. Only if a probe is converted to a highly de-esterified hydrophilic species, such as the salt form, will inhibition of permeability result. Low concentration or low activity of cytosolic nonspecific esterase will always be problematic for detecting cytosolic calcium with calcium probes. In the extreme, as presented here, some cells may show no calcium signal. If hydrolysis is slow, lipophilic ester or partially de-esterified species will be present in the cytosol; consequently, probe accumulation in other organelles will be likely. Compartmentalization also is favored by extending incubation times or by raising the concentration of the probe. Problem avoidance and prediction of possible compartmentalization Even if the general model described above is valid and of practical use to some, most laboratory workers would find it too cumbersome to be practical for applying the specifics of calcium probe compartmentalization. What is needed is some more algorithmic scheme for predicting compartmentalization and for avoiding trouble. Such a scheme is presented as a chart in Fig. 7.

It must be appreciated, however, that this chart is only a guide to what can happen with a particular probe. Whether the worst that can happen does happen is determined in part by the other factors described, namely, probe characteristics and concentration, incubation time and temperature, and especially the cell lines with their variable esterase concentrations. Assessing the inclusive model The algorithmic chart (Fig. 7) also facilitates assessment of the underlying general model and allows two questions to be asked that bear on validity. First, does the scheme predict correctly compartmentalizations reported by bench workers for the common cytosolic calcium probes? Second, does the scheme predict the localization patterns seen with organelle-specific, i.e., non-cytosolic, calcium probes? The first question can be addressed by considering the 25 specific predictions regarding probe accumulation sites listed in column four of Table 1. Comparison of these predictions with the reportsdescribed in Table 4 indicates that intracellular localization sites were predicted correctly two thirds of the time. Moreover, all errors involved predicted accumulations that were not observed in the cited reports. Because Table 4 is not an exhaustive listing of all reports of compartmentalization for the named probes, however, the one third negative predictive error doubtless is pessimistic. We may therefore regard the validity of the general model as being supported. Note that if accumulation in endoplasmic reticulum is considered (the reasons for its exclusion were noted above), the predictive accuracy remains unchanged. To address the second question, we considered probes that are intended to detect calcium ions within mitochondria, namely, compounds intended to be compartmentalized in such organelles (Davidson and Duchen 2012). The numerical structure parameters for the AM esters of six such compounds (rhod-5N, rhod-FF, X-rhod-1, X-rhod-5F, X-rhodFF, X-rhod-5N) were estimated and algorithm in Fig. 6 was applied. This predicted mitochondrial compartmentalization for all six probes, which also supports the underlying general model. Conclusions We describe here a general model to aid the understanding and practical application of calcium probes. We emphasized the following practical



Fig. 6. Simplified graphic scheme illustrating a general model of the interactions of AM ester calcium probes with a generic live cell of which the simple model is a subset.

issues: 1) Super lipophilic AM ester probes, e.g., calcium orange, fluo-3, fura red, are not membrane permeable; consequently easy entry into cells requires the presence of SA in the incubation medium 480

or esterase in the plasma membrane or both. 2) Obtaining cytosolic calcium signals requires sufficient cytosolic esterase, which varies considerably both among cell lines and among cell populations


List structure parameters of AM ester

Specify staining protocol

N

Is SA present?

Y


Start

Inspect Fig 2 for chemical species resulting from extracellular ester hydrolysis

Answer questions for ALL chemical species present

Y

Is species membrane permeant?

Y

N Y

plasma membrane uptake?

Y Endo/ lysosomes

N List structure parameters of all species: cf Table 1

Pinocytosis occurs?

Y

endo/lysosome uptake?

Y N

mitochondrial uptake?

Mitochondria

Y Ester hydrolysed?

Inspect Fig 2 for chemical species from intracellular ester hydrolysis; list their structure parameters, cf Table 1

Cytosol

Y

Membrane internalised?

Plasma membrane

Y N

nuclear uptake?

Nucleus

Y

Activity step

Organelle contains esterase in many cell lines See Table 2 for information needed to answer query

Decision

Compartmentalisation or uptake site

N Retained in

Cytosol

See Table 3 for information needed to answer query

Fig. 7. Flow chart predicting occurrence or non-occurrence of compartmentalization of AM ester calcium probes in living cells using their structure parameters plus appropriate QSAR decision rule models. a/c accumulation.

of a single cell line. 3) Presence of unhydrolyzed esters in the cytosol owing to low cytosolic esterase concentration or activity, especially when long incubation times are used, or high extracellular probe concentration favors compartmentalization. We created an algorithmic chart in graphic form to assess possible compartmentalization and guides to relevant QSAR models with notes on estimation of the structured parameters required for insertion in these models.

Acknowledgments R.W.H. thanks Dr. R. Aitken, School of Life Sciences, College of Medical, Veterinary and Life Science, Glasgow University, Scotland UK for providing facilities. K.T and P.D are indebted to a HEA PRTLI4 Grant to Fund the National Biophotonics and Imaging Platform Ireland (www.nbipireland.ie) and IRCSET Enterprise Partnership Scheme in conjunction with Hewlett-Packard Galway Ltd.


Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.


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