Iozzo Renato V. Proteoglycan Protocols

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428 Moscatello and Iozzo

promiscuous binding properties, establishing a direct interaction of proteoglycans with

receptor tyrosine kinases requires a number of methodological approaches. Examples

of a few such techniques are presented here, but the reader is encouraged to consider

other chapters in this volume as well, since other approaches may be useful in the

context of different ligand–receptor systems. While the sample protocols presented all

involve the EGFR and decorin, the same approaches should prove useful for other

receptor–proteoglycan interactions.

2. Materials

2.1. Cells, Culture Media, and Growth Factors

1. A431, HT-1080, and a wide variety of other human tumor cell lines can be obtained from

the American Type Culture Collection (Rockville, MD), as can CHO and NIH-3T3 cells.

The latter two cell lines are widely used for transfection and overexpression of proteins of

interest.

2. Fetal bovine serum (FBS) is used at 10% (v/v) to culture A431 and most human

tumor cell lines. Ten percent calf serum (CS) is used to grow NIH-3T3 cells and

transfectants. Both can be obtained from Hyclone (Logan, UT) or Life Technologies

(Rockville, MD). A431 and 3T3 cells are grown in Dulbecco’s modified Eagle

medium (Life Technologies or Mediatech) (Herndon, VA). RPMI 1640, D-PBS,

epidermal growth factor, and trypsin (0.25% for A431, 0.05% for 3T3) are all obtained

from Life Technologies.

3. Conditioned media for soluble receptor ectodomain or recombinant proteoglycan produc-

tion are readily concentrated using Amicon Ultrafree-15 centrifugal filter units with Biomax-

50 membranes (50,000-dalton nominal molecular weight limit) and filter-sterilized with

0.45-µm pore vacuum filter units (Millipore, Bedford, MA).

2.2 Antibodies and Biochemical Reagents

1. Anti-EGFR and anti-phosphotyrosine antibodies can be obtained from Promega

(Madison, WI) and Upstate Biotechnology (Lake Placid, NY), respectively. Antibod-

ies to a variety of EGFR epitopes are also available from Calbiochem-Novabiochem

Corp. (San Diego, CA).

2. Bovine serum albumin (BSA, Cohn fraction V) and all other reagents not specified are from

Sigma Chemical (St. Louis, MO).

a. For blocking cells, a 0.2% BSA (w/v) solution in RPMI 1640 is filter sterilized and

stored at 4°C.

b. Membranes are blocked with 2% BSA in TBST (TBS [100 mM Tris-HCl, pH 7.5, 150

mM NaCl] plus 0.1% Tween 20) or 5% nonfat dry milk in TBST.

c. Kits for labeling by the Iodo-Gen method are available from Pierce Chemical (Rock-

ford, IL). [

125

I]NaI is from Amersham (Arlington Heights, IL).

d. Active, affinity-purified EGFR was purchased from Sigma Chemical (cat. no. E2645).

[γ-

P] ATP (6000 Ci/mmol), enhanced chemiluminescence (ECL) reagents and

Hybond ECL membranes were from Amersham. Nitrocellulose membranes from

Schleicher & Schuell (Keene, NH) also work well.

e. Immulon 4HXB wells are from Dynex Technologies (Chantilly, VA).

f. The membrane-impermeable crosslinker bis[sulfosuccinimidyl]-suberate (BS

) is

from Pierce. Nickel-nitrilo-triacetic acid (Ni-NTA) columns for purification of His

tagged recombinant proteins are from Qiagen (Valencia, CA).

Interaction with RTKs 429

3. PBS/TDS: 10 mM Na

HPO

, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate,

0.1% sodium dodecyl sulfate, 0.02% sodium azide, 0.004% sodium fluoride, and

1 mM sodium orthovanadate, pH 7.25. The stock is stored at 4°C, and phenylmethylsulfonyl

fluoride (made as a 100× stock in isopropanol and stored at –20°C) is added to 100 µg/mL

immediately before use.

4. In vitro reactions with EGFR kinase are performed in 20 mM HEPES, pH 7.4, 2 mM

MnCl

, 10 mMp-nitrophenyl phosphate, 40 mM Na

, 0.01% BSA with 15 mM ATP

(kinase buffer). For blocking wells in radioligand binding assays, 0.1% BSA in TBS with

2 mM CaCl

, 2 mM MgCl

, and 0.02% NaN

is used.

3. Methods

3.1. Binding Competition with Known Receptor Ligands

1. Growth of cells. Cells are seeded into 24-well (16-mm) plates and grown for 2 d or until

just confluent. Sufficient wells must be plated for triplicate determinations of all condi-

tions. A431 cells are commonly used to analyze binding to the EGFR.

2. Recombinant human decorin (see Note 1) is labeled to high specific activity (~8 × 10

cpm/mg) with [

125

I]NaI (Amersham) by the Iodo-Gen method (Pierce Chemical).

3. The cells are washed twice with D-PBS, then blocked with 1 mL of RPMI-1640 containing

0.2% BSA at 4°C or on ice for 1 h (see Note 2).

4. The blocking medium is removed, and the cells are then incubated with 0.5 mL of 0.2%

BSA/ RPMI-1640 containing ca. 200 ng (1.5 × 10

cpm)

125

I-decorin with or without the

appropriate concentrations of specific ligand. In this example, EGF is added at concentrations

from 10 to 300 ng/mL. A 100-fold excess of unlabeled test ligand is added to triplicate wells

to correct for background binding.

5. The binding media are removed, and the wells are washed three times with cold PBS. The

cells are then dissolved in 1 mL of 1 M NaOH and the radioactivity measured in a gamma

counter. The means ± SD of the triplicate determinations are then calculated.

3.2. Chemical Crosslinking to Receptors

1. A431 cells (or other cells of interest) are plated in 6-well (35-mm) plates and grown until

nearly confluent. The cells are incubated in serum-free medium (e.g., DMEM only) for

24–36 h.

2. The cells are washed twice with D-PBS, then blocked with 1 mL of RPMI-1640 containing

0.2% BSA at 4°C or on ice for 1 h.

3. The cells are incubated with decorin (DCN), decorin protein core (∆DCN)(100 µg/mL), or

EGF (16 nM), or combinations thereof, at 4°C or on ice for 1 h.

4. The cells are washed three times with 2 mL of cold PBS per well and incubated for 10 min

at room temperature in PBS with 15 mM crosslinker BS

. If the lysates are to be subjected

to immunoprecipitation, the cells are scraped into 0.5 mL/well ice-cold lysis buffer, centri-

fuged at 12,000g in microcentrifuge tubes for 10 min at 4°C, and the supernatants are

transferred to clean tubes. If the lysates are only to be analyzed by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE), the cells may be lysed directly in SDS

sample buffer. The cell lysates are separated by SDS-PAGE in thin (0.75-mm), 3–15%

acrylamide gels, transferred to nitrocellulose membranes, and subjected to Western

immunoblotting with anti-EGFR antibody (18).

5. After exposing the blot to film, the autoradiogram is analyzed for the presence of receptor

monomers and dimers; the latter should be present only in lanes with crosslinker. In the case

of studies involving the EGFR, the monomeric receptor migrates at an M

of 170 kDa,

430 Moscatello and Iozzo

and the dimers at an M

of 340 kDa. Slower-migrating species may also be observed if the

ligand is also crosslinked; in the present instance, a single decorin molecule bound to a

EGFR dimer would be expected to migrate at an M

of ~440 kDa, and two decorin

molecules per dimer would be expected to migrate at an M

of ~540 kDa (see Note 3).

3.3. Interaction with Soluble Receptor Ectodomain

1. A431 cells synthesize a secreted form of EGFR of ~105 kDa lacking the transmembrane and

intracytoplasmic domains (19). A431 cells are grown to near confluence in standard medium,

washed twice with D-PBS, and incubated with serum-free medium for 24–48 h (see Note 4).

2. Medium conditioned by confluent A431 cells is concentrated by centrifugation in

Ultrafree-15 (50,000-nmwl) units at 4°C according to the manufacturer’s protocol. The

conditioned medium is then filter-sterilized (0.45-µm-pore filters) or centrifuged at high

speed to remove insoluble material. NaN

(0.02% final concentration) may be added to

inhibit microbial growth. Such preparations are ideally used fresh, but may be stored for

a few days at 4°C, or mixed with an equal volume of glycerol and stored at –20°C.

3. Serial dilutions of BSA, decorin or its protein core are slot- or dot-blotted onto nitrocellu-

lose membranes using a vacuum manifold and blocked overnight at 4°C (or for 1 h at room

temperature) with 5% FBS and 5% nonfat milk (see Note 5).

4. The blots are washed several times in TBST and incubated with the serum-free medium

conditioned by the A431 cells. The blots are again washed three times, then incubated

with gentle agitation using an antibody against the ectodomain of the human EGFR

(e.g., anti-LEEKK of the human EGFR N-terminal sequence, affinity purified on pep-

tide linked to Sepharose) at 1 µg/mL in BSA/TBST for 1–2 h at room temperature. The

antibody used should not interfere with the binding, in this case because it recognizes

the N-terminal end of the EGF receptor, distant from the known ligand-binding site. It

is desirable, however, to use an antibody that does recognize the ligand-binding domain

as an additional control (e.g., monoclonal antibody 225 raised against the EGF-binding

domain of the EGFR, ref. 20).

5. The membranes are washed three times with TBST and incubated with the appropriate

secondary antibody (against either rabbit polyclonal or mouse monoclonal antibody) for 1 h

at room temperature. Alternatively, radiolabeled secondary antibodies may be used.

6. Finally, the membranes are incubated with anti-rabbit or anti-mouse IgG coupled to horse-

radish peroxidase (1/5000) in TBS-T, washed three times, and incubated with enhanced

chemiluminescence reagent. Multiple exposures are performed to guarantee linearity, and

the intensities of the bands are quantitated by laser scanning densitometry.

3.4. In Vitro Phosphorylation of the Receptor Tyrosine Kinase

1. Constant amounts (~300 ng) of immunopurified EGFR are incubated with kinase buffer

alone or containing various concentrations (5–20 µg) of decorin, decorin protein core, or

collagen type I. A control tube should include 100 ng of EGF.

2. After 15 min of incubation in kinase buffer, 1 µCi [γ-

P]ATP and 0.2% NP40 are added in

a final volume of 60 µL. The mixtures are incubated for an additional 10 min, and the

reactions are terminated by boiling in SDS sample buffer.

3. The samples are separated by SDS-PAGE, and the gel is either dried, or the proteins are

transferred to nitrocellulose. The latter technique enables one to use immunoblotting to

confirm the identities of the labeled proteins. Phosphorylated proteins are visualized by

autoradiography. Control samples omitting either [γ-

P]ATP or EGFR should show no

activity (see Fig. 1).

Interaction with RTKs 431

3.5. Interaction of Proteoglycan with Purified Receptor Kinase

1. Column protocol: Recombinant decorin with an N-terminal tag of six histidine residues

(6 His) allows for a rapid and efficient purification via Ni-NTA affinity chromatography (21).

2. Purified EGFR is phosphorylated to a high specific activity using the EGF control reac-

tion in vitro phosphorylation procedure described in Subheading 3.4., steps 1 and 2,

except that the reactions are not boiled in sample buffer and labeled receptor is separated

from unincorporated label by Sephadex G-50 chromatography (Roche Quick-spin

columns).

3. Constant amounts of

P-labeled EGFR are incubated with increasing concentrations of

decorin, decorin core protein, or control proteins for 30 min at 4°C under gentle agita-

tion. The spin columns are equilibrated with three column volumes of binding buffer

(300 mM NaH

, pH 8.0, 300 mM NaCl), and then the samples are applied and spun

at 750 g for 5 min.

4. Following two consecutive washes, the bound decorin/EGFR complexes are eluted with

buffer containing 250 mM imidazole. Aliquots of the fractions are counted in a scintilla-

tion counter and the remainder are analyzed by SDS-PAGE and autoradiography.

5. Solid-phase binding protocol: Immulon 4HXB wells are coated with 1 µg (~22 pmol) of

recombinant decorin in 100 µL of PBS for 2 h at 37°C or overnight at 4°C. The wells are

washed three times with PBS and blocked with 0.1% BSA in TBS supplemented with

2mM CaCl

, 2 mM MgCl

, and 0.02% NaN

(blocking buffer) for 1 h.

6. Purified EGFR is labeled as described in step 2, 0.1–10 pmol

P-EGFR is added in block-

ing buffer, and the wells are incubated under gentle shaking (60 rpm) for 4–14 h at 4°C.

Fig. 1. In vitro phosphorylation assay using immunopurified EGFR and decorin, its protein

core, or collagen. Constant amounts (~300 ng) of immunopurified EGFR were preincubated with

buffer alone (lane 1) or containing decorin (lanes 2–4; at 5, 10, and 20 µg, respectively), decorin

core protein (lanes 5–7; at 5, 10, and 20 µg, respectively), or collagen (lanes 8–10; at 5, 10, and 20

µg, respectively). After 15 min in kinase buffer, 1 µCi of [γ-

P]ATP and 0.2% Nonidet P-40

were added. The mixtures were incubated for an additional 10 min, stopped by boiling in SDS

buffer, and analyzed by SDS-PAGE and autoradiography. Note the strong EGFR

autophosphorylation induced by decorin and decorin core protein relative to the collagen control.

432 Moscatello and Iozzo

Background binding is corrected for by incubation with at least 100-fold molar excess

unlabeled EGFR. All samples should be done in triplicate. After incubation the wells are

washed three times with ice-cold TBST and measured using a scintillation counter. Data

are analyzed by Scatchard analysis, e.g., using the Ligand program (18).

4. Notes

1. It is imperative that the proteoglycans in question be purified by techniques that permit

retention of a “native” conformation (21). Many commercially available extracellular

matrix proteins and proteoglycans, particularly those from tissues such as skin or tendon,

are prepared by harsh extraction methods that result in denatured products. Not surpris-

ingly, such preparations have greatly reduced or no biological activity (18,22), and of

course are unsuitable for use in studies of signaling interactions. Recombinant or endog-

enous materials isolated from cell cultures are generally suitable, as they can be purified

by relatively gentle procedures (see Chaps. 1, 4, 20, 21; and ref. 21 for relevant proto-

cols). Wherever possible, it is also desirable to use a closely related proteoglycan as a

control for nonspecific binding. For example, we use biglycan (17,21) as a control for

decorin in experiments with EGFR; biglycan does not bind or activate the EGFR (17,22).

We also use a deglycosylated proteoglycan, or mutated recombinant protein lacking the

glycosaminoglycan chain (17,21,23), to ascertain the importance of the glycosaminogly-

can chain in the interaction.

2. Only purified proteins such as bovine serum albumin should be used as blocking agents in

binding experiments. Gelatin cannot be used, as it is composed of collagen, with which

many proteoglycans can interact. Other commonly used blocking agents such as nonfat milk

or sera contain growth factors or matrix components that may interact with either the

receptor or proteoglycan of interest, respectively.

3. Very large complexes (>400 kDa) do not penetrate standard polyacrylamide gels, and are

not efficiently transferred to membranes for blotting. A possible alternative approach would

be the use of cleavable crosslinkers such as 3,3'-dithiobis[sulfosuccinimidyl] propionate

(Pierce). Crosslinked receptor–proteoglycan complexes are immunoprecipitated from

lysates, the cleaving reagent is added, the samples are boiled in SDS sample buffer, sepa-

rated by SDS-PAGE, and analyzed by immunoblotting against both the receptor and the

proteoglycan (or labeled ligand may be used).

4. Alternatively, constructs encoding ectodomains of other receptors of interest may be trans-

fected into NIH-3T3 cells by standard methods. Conditioned media would be produced and

collected in the same fashion as for EGFR ectodomain from A431, although the kinetics of

expression would have to be determined empirically. Expression of the soluble receptor

would be confirmed by immunoblotting, and an epitope tag such as 6 ↔ His would provide

a convenient “handle” for purification and isolation of receptor–ligand complexes.

5. Alternatively, the reciprocal experiment may be performed; that is, the soluble receptor

preparation may be slot-blotted and incubated with proteoglycan solution. The blots would

then be probed with antibody to the proteoglycan, or labeled proteoglycan could be used.

References

1. Iozzo, R. V. and Murdoch, A. D. (1996) Proteoglycans of the extracellular environment:

clues from the gene and protein side offer novel perspectives in molecular diversity and

function. FASEB J. 10, 598–614.

2. Somasundaram, R. and Schuppan, D. (1996) Type I, II, III, IV, V, and VI collagens serve as

extracelular ligands for the isoforms of platelet-derived growth factor (AA, BB, and AB).

J. Biol. Chem. 271(43), 26884–26891.

Interaction with RTKs 433

3. Iozzo, R. V. (1997) The family of the small leucine-rich proteoglycans: key regulators of

matrix assembly and cellular growth. Crit. Rev. Biochem. Mol. Biol. 32, 141–174.

4. Vogel, W., Gish, G. D., Alves, F., and Pawson T. (1997) The discoidin domain receptor

tyrosine kinases are activated by collagen. Mol. Cell. 1,13–23, 1997.

5. Shrivastava, A., Radziejewski, C., Campbell, E., Kovac, L., McGlynn, M., Ryan, T. E.,

Davis, S., Goldfarb, M. P., Glass, D. J., Lemke, G., and Yancopoulos G. D. (1997) An

orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen

receptors. Mol. Cell. 1, 25–34.

6. Krusius, T. and Ruoslahti, E. (1986) Primary structure of an extracellular matrix proteoglycan

core protein deduced from cloned cDNA. Proc. Natl. Acad. Sci. (USA). 83, 7683–7687.

7. Day, A. A., McQuillan, C. I., Termine, J. D., and Young, M. R. (1987) Molecular cloning

and sequence analysis of the cDNA for small proteoglycan II of bovine bone. Biochem. J.

248, 801–805.

8. Fisher, L. W., Termine, J. D., and Young, M. F. (1989) Deduced protein sequence of bone

small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and sev-

eral nonconnective tissue proteins in a variety of species. J. Biol. Chem. 264, 4571–4576.

9. Toole, B. P. and Lowther, D. A. (1968) The effect of chondroitin sulphate-protein in the

formation of collagen fibrils in vitro. Biochem. J. 109, 857–866.

10. Vogel, K. G., Paulsson, M., and Heinegård, D. (1984) Specific inhibition of type I and

type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem. J. 223,

587–597.

11. Adany, R., Heimer, R., Caterson, B., Sorrell, J. M., and Iozzo. R. V. (1990) Altered

expression of chondroitin sulfate proteoglycan in the stroma of human colon carcinoma.

Hypomethylation of PG-40 gene correlates with increased PG-40 content and mRNA lev-

els. J. Biol. Chem. 265, 11389–11396.

12. Santra, M., Skorski, T., Calabretta, B., Lattime, E. C., and Iozzo, R. V. (1995) De novo

decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

Proc. Natl. Acad. Sci. (USA) 92, 7016–7020.

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(1997) Ectopic expression of decorin protein core causes a generalized growth suppres-

sion in neoplastic cells of various histogenetic origin and requires endogenous p21, an

inhibitor of cyclin-dependent kinases. J. Clin. Invest. 100, 149–157.

14. De Luca, A., Santra, M., Baldi, A., Giordano, A., and Iozzo, R. V. (1996) Decorin-induced

growth suppression is associated with upregulation of p21, an inhibitor of cyclin-depen-

dent kinases. J. Biol. Chem. 271, 18961–18965.

15. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) The p21

Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell.

75, 805–816.

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D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) WAF1, a potential mediator of

p53 tumor suppression. Cell. 75, 817–825.

17. Moscatello, D. K., Santra, M., Mann, D. M., McQuillan, D. J., Wong, A. J., and Iozzo,

R. V. (1998) Decorin suppresses tumor cell growth by activating the epidermal growth

factor receptor. J. Clin. Invest. 101, 406–412.

18. Iozzo, R. V., Moscatello, D. K., McQuillan, D. J., and Eichstetter, I. (1999) Decorin

is a biological ligand for the epidermal growth factor receptor. J. Biol. Chem. 274,

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434 Moscatello and Iozzo

19. Weber, W., Gill, G. G., and Spiess, J. (1984) Production of an epidermal growth factor

receptor-related protein. Science 224, 294–297.

20. Fan, Z., Lu, Y., Wu, X., and Mendelsohn, J. (1994) Antibody-induced epidermal growth

factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squa-

mous carcinoma cells J. Biol. Chem. 269, 27595–27602.

21. Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) Recombinant decorin

glycoforms. Purification and structure. J. Biol. Chem. 271, 19578–19584.

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5317–5323.

Principles of Ca

Imaging 435

435

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Measuring Single-Cell Cytosolic Ca

Concentration

in Response to Proteoglycans

Sandip Patel and Renato V. Iozzo

1. Introduction

is a crucial biological messenger involved in a host of diverse cellular

processes (1). Many hormones, growth factors, and neurotransmitters raise cytosolic

levels through activation of phospholipase C, which catalyzes the production of

the second messenger, inositol 1,4,5-trisphosphate (IP

) (2). G-protein-linked recep-

tors and receptor tyrosine kinases couple to distinct isoforms of phospholipase C (3).

Once generated, IP

raises cytosolic Ca

levels by activating Ca

channels located on

the membranes of intracellular Ca

stores (4,5).

Extracellular matrix constituents are also known to induce Ca

signals through

activation of integrins (6). More recently, we have demonstrated that decorin, a

member of the small leucine-rich proteoglycan family (7), mediates cytosolic Ca

increases through a novel action on the epidermal growth factor (EGF) receptor

(Fig. 1) (8). Indeed, interaction of the extracellular matrix with cell surface receptor

tyrosine kinases may be more widespread than previously thought (9–11). Changes in

cytosolic [Ca

] are therefore likely to be important in mediating the effects of the

extracellular matrix on cell function. In this chapter, the principles of measuring single

cell cytosolic [Ca

] are discussed.

Measuring cytosolic [Ca

] at the single cell level using imaging approaches has

several advantages over monitoring [Ca

] in populations of cells. In many cells,

submaximal hormone stimulation induces complex changes in cytosolic [Ca

including Ca

oscillations (12), which in population studies are “averaged” out and

remain undetected. Such temporal averaging also distorts the kinetics of even relatively

simple changes in cytosolic [Ca

]. For example, if all cells do not respond under a

particular condition or do so with differing latencies, the average population response

will underestimate the peak and rate of rise in cytosolic [Ca

] of the responsive cells.

In addition, population studies provide no spatial information concerning the Ca

436 Patel and Iozzo

Fig. 1. Decorin mediates increases in cytosolic [Ca

] of single A431 cells. Images of single fura-2-loaded A431

cells before (top left) and after (at the times indicated) stimulation with decorin (100 µg/mL). The overlaid red image is

proportional to changes in [Ca

]. Bottom right-hand panel shows a typical [Ca

] response of a single cell.

436

Principles of Ca

Imaging 437

increase within the cell. Indeed, Ca

signals often originate from a specific cellular

locus and travel throughout the cytosol as planar or even spiral “waves” (12). Finally,

population measurements do not distinguish signals from damaged or contaminat-

ing cells.

1.1. Fluorescent Ca

-Sensitive Indicators

The use of fluorescent Ca

-sensitive indicators is currently the most popular

method for monitoring cytosolic [Ca

]. Fluorescence is the process whereby a mol-

ecule in an excited state dissipates some of its energy prior to relaxation, upon absorb-

ing a photon of light, such that the emitted light is of lower energy and thus longer

wavelength than that of the exciting light. Fluorescent Ca

indicators are molecules

that change their fluorescence properties upon binding Ca

, most of which are based

on the Ca

chelators, ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) and

1,2-bis(2-Aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA). Many Ca

indicators are commercially available for measuring [Ca

] [see ref. (13)]. Factors to

be considered when choosing a dye are its molar extinction coefficient and quantum

yield—that is, the efficiency of light absorption and the amount of light emitted rela-

tive to what was absorbed. In order to maximize fluorescence signals in response to

, a dye with an affinity appropriate to the range of anticipated [Ca

] changes

should be chosen. Upon stimulation, cytosolic [Ca

] can be elevated several fold from

resting levels of < 200 nM.

-sensitive indicators can be classified according to the nature of the fluores-

cence change that Ca

binding induces. Ratiometric (dual-wavelength) indicators

undergo a shift in their fluorescence spectra (excitation or emission) upon binding

, whereas intensiometric (single wavelength) dyes do not. Fura-2 (14) is a

ratiometric indicator that is currently the most commonly used Ca

-sensitive dye in

imaging studies (Fig. 2A). Important properties of this dye include the following:

1. In the absence of Ca

, the excitation and emission peaks are 360 and 510 nm, respectively.

2. In the presence of saturating Ca

levels, the excitation peak is shifted to 340 nm with no

appreciable change in the emission spectra.

3. The isosbestic (Ca

-independent) wavelength is 360 nm.

4. Reciprocal changes in the fluorescence of the dye occur upon Ca

binding when excited

either side of the isosbestic wavelength.

5. At 340-nm excitation, fluorescence intensity increase as the [Ca

] increases.

6. At 380-nm excitation, fluorescence intensity decreases as the [Ca

] increases.

7. The ratio of the two intensities at 340 and 380 nm is proportional to [Ca

Fluo-3 is an example of an intensiometric indicator. This visible wavelength dye

has excitation and emission peaks of ~500 and ~525 nm, respectively. This dye is

particular useful in that in it undergoes a large increase in fluorescence upon binding

. Additionally, the absolute florescence of the Ca

-free dye is extremely low,

thus further improving the signal-to-noise ratio. Ratiometric dyes, however, are

advantageous over intensiometric dyes in several respects. For intensiometric

indicators, fluorescence is proportional to the concentration of both Ca

and the dye.

Since changes in cellular dye concentration can occur during experimentation, such