Coumarin-phalloidin: a new actin probe permitting triple

immunofluorescence microscopy of the cytoskeleton

J. V. SMALL1, S. ZOBELEY2, G. RINNERTHALER1 and H. FAULSTICH2

'institute of Molecular Biology of the Austrian Academy of Sciences, Billrothstr. 11, 5020 Salzburg, AustriazMax-IJlanck-lnstitut fur Medizinische Forschung, Abteihing Physiologie, Jahnstr. 29, Heidelberg 1, D-6900 FRC

Summary

7 - Diethylamino - 3 - (4 - isothiocyanotophenyl) - 4 -methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a newphalloidin derivative, coumarin-phalloidin, flu-orescent in the blue region of the spectrum.Coumarin-phalloidin binds to actin with around100-fold less affinity than unconjugated phal-loidin, but with enough avidity to make it a useful

stain for actin filaments. Appropriate filter combi-nations permit triple immunofluorescence mi-croscopy of the cytoskeleton -with fluorescein andrhodamine conjugates together with coumarin-phalloidin.

Key words: coumarin-phalloidin, actin,immunofluorescence microscopy.

Introduction

The mode of involvement of the cytoskeleton in diversecellular functions is an area of central interest incontemporary cell biology. Mediation of function isnow generally presumed to involve an ever increasingnumber of proteins that are associated with the threefibrillar systems of the cell: the actin filaments, micro-tubules and intermediate filaments (Pollard & Cooper,1986; Traub, 1985; Soifer, 1986). An integral part ofstudies on these associated proteins is their localizationin the cytoskeleton, in one or more of the fibrillarsystems, and for this purpose standard immunologicaland other probes for the primary fibrillar proteins arenow commercially available and in general use. How-ever, localization studies have been restricted to doublefluorescence microscopy, using the most commonlyavailable fluorescein and rhodamine-labelled conju-gates. Among these, different phalloidin analogues,emitting in the red (Wieland et al. 1983; Faulstich et al.1983)orgreen(Wulf^a/. 1979; Barak&Yocum, 1981)have proved invaluable as markers of the actin system.

In the course of our recent studies on fibroblastlocomotion it became necessary to visualize actin inaddition to two other cytoskeletal components (vincu-lin and tubulin) and for this purpose a compromiseprocedure was followed that involved overstaining oneof the first two labels (after photography) with a third

Journal of Cell Science 89, 21-24 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

label carrying the same fluorophore (Small & Rinner-thaler, 1985; G. Rinnerthaler, B. Geiger & J. V. Small,unpublished data). This experience prompted us toconsider the employment, instead, of a third, bluefluorescent probe. In an earlier study, Namihisa et al.(1980) had indeed described the use of such a probe, athiol-reactive coumarin derivative (Machida et al.1975), on heavy meromyosin or myosin subfragment-1for the demonstration of actin in frozen sections. Since,however, myosin subfragments are not very stable inthe long term the availability of this probe relies on aready supply of the appropriate subfragments. In analternative approach we have tested the possibility ofcoupling coumarin to phalloidin and here describeresults obtained with the most successful of the deriva-tives synthesized: 7-diethylamino-3-(4-isothiocyanato-phenyl)-4-methylcoumarin (CPITC)-phalloidin. Bythe use of commercially available filters, tripleimmunofluorescence microscopy is readily achievedwith this reagent in combination with fluorescein- andrhodamine-labelled probes.

Materials and methods

Preparation of coiimarin-labelled phalloidinCoumarin-labelled phalloidin (A'/r 1240) was preparedby a method similar to that used for tetramethylrhodaminylphalloidin (Faulstich et al. 1983).

21

A 10 ing sample of aminomethyldithiolanophalloidin hy-drochloride (Wieland et al. 1980) was dissolved in 1-5 ml70% dimethylsulphoxide. The pH was adjusted to 8-5 withlM-NaOH, and lOmg 7-diethylamino-3-(4-isothiocyanato-phenyl)-4-methylcoumarin (D-347, Molecular Probes) dis-solved in 1 ml dimethylsulphoxide was added. After reactingthe mixture for 12 h at 4°C, the solvent was removed in vacuoand the residue dissolved in methanol. The solution wasapplied on two precoated thin-layer chromatography (tic)plates (20cmX20 cm, SIL1CIA GEL 60 F-254, Merck,Darmstadt) and developed with chloroform/NH3-saturatedmethanol/water (65:25:4 by vol.). The product appeared asthree bands with greenish fluorescence (RF+ 0-6-0-7),which were scraped off and eluted with methanol. After finalpurification on a small column (15 cm X 1 cm) with SephadexLH20 (Pharmacia, Uppsala) eluted with methanol, coumarin-labelled phalloidin (7-6 mg, 55 %) was obtained as a mixtureof 3-4 isomers. Coumarin-phalloidin was stored after dryingin vacuo or as a stock of 0-1 mgml"1 in methanol, at —20°C(coumarin-phalloidin will be distributed by SIGMA Chem.Co.)

Two other coumarin reagents were successfully coupledto aminomethyldithiolanophalloidin, 7-((4-chloro-6-diethyl-amino)-s-triazin-2-yl)amino)-3-phenylcoumarin (C-192,Molecular Probes), and 7-methoxycoumarin-4-acetic acid(M-188. Molecular Probes). However, the affinity of thesederivatives for actin filaments was poor, or non-existent.

Beside the coumarin fluorophore reagents, other blue-fluorescent chromophores were tested; among them stilbene,anthracene, acridine and pyrene derivatives. Specifically,these were: 4-acetamido-4-isothiocyanatostilbene-2,2-disul-phonic acid (A-339, Molecular Probes, SITS); 2-anthracene-sulphonyl chloride (A-448, Molecular Probes); succinimidylacridine-9-carboxylate (S-1127, Molecular Probes); succini-midyl 1-pyrenebutyrate (S-130, Molecular Probes). All thesereagents yielded the corresponding phalloidin derivatives butwere not investigated further because they were foundunsuitable as actin stains.

Spectral characteristics and actin bindingAbsorption and fluorescence spectra of coumarin-phalloidinwere recorded in water using a Beckman 25 spectropho-tometer and a Perkin Elmer MPF-3 fluorescence spectropho-tometer, respectively.

The affinity of binding of coumarin-phalloidin to actinwas carried out according to a procedure to be fully described(Faulstich et al. unpublished). In brief, the assay relied onthe measurement of displacement of unconjugated, tritiated[ H]phalloidin from F-actin filaments by progressivelyhigher concentrations of coumarin-phalloidin (see Fig. 1).

Fixation and labelling proceduresHuman skin fibroblasts (provided by Dr M. Malecki, War-saw), plated on 10 mm diameter glass coverslips, were fixedfor 2 min at room temperature in a glutaraldehyde/Triton X-100 mixture (0-25 % glutaraldehyde/0-3 % Triton), followedby 10 min in 1 % glutaraldehyde, both made up in a cytoskel-eton buffer (see Small el al. 1986). They were then treatedwith NaBH4. (Weber et al. 1978), 0-Smgml"1 in ice-coldcytoskeleton buffer (three times, 5 min) prior to immuno-labelling. The coverslips were incubated in the first antibody

0-2ccjo

I 0-1reaX

100 1000 10000Phalloidin concentration (X10~8M)

Fig. 1. Affinity of coumarin-phalloidin for F-actin.Curves show amount of tritiated phalloidin displaced fromF-actin by increasing concentrations of, respectively,unconjugated phalloidin (A A) and coumarin-phalloidin ( • • ) . The data indicate a 125-fold loweraffinity of coumarin-phalloidin for F-actin, as compared tounconjugated phalloidin.

mixture containing mouse anti-vimentin (a gift from Dr S.Blose) and rabbit anti-tubulin (a gift from Dr J. De Mey)diluted in a Tris-buffered saline (GB, see Small et al. 1986)with added 1 % bovine serum albumin (BSA) and 2 % normalgoat serum, for 40 min at room temperature, on parafilm.After washing (twice, 10 min) in GB they were transferred tosheep anti-mouse biotin (Amersham International, UK) inGB containing 1 % BSA for 30 min at room temperature andthen, after a further wash, to a mixture of goat anti-rabbitrhodamine (a gift from Dr B. Geiger), streptavidin-FITC(Amersham International) and CPITC-phalloidin, alldiluted in GB containing 1 % BSA. The CPITC-phalloidinwas used at a concentration of 1—2/igml~' (1-2 ,UM). The lastincubation of reagents was carried out on ground-glass slidesinstead of parafilm, since the latter produced detrimentalstaining with phalloidin derivatives. After the final wash, thecoverslips were mounted in Gelvatol (20-30, MonsantoCorp., NY) made up, as described for Elvanol by Rodriguez& Deinhardt (1968), and with added w-propyl gallate (Giloh& Sedat, 1983), l,4-diazobicyclo-(2,2,2)-octane (DABCO)or phenylenediamine (Johnson et al. 1982) as anti-bleachagents. Effective concentrations of additives were2-Smgmr 1 for re-propyl gallate, lOOmgml"1 for DABCO(Langangere^a/. 1983) and 1 rngmP' for phenylenediamine.

Light microscopyPreparations were observed and photographed on a Zeissphotomicroscope III fitted with an epi-condensor and sidemounted 50 W Hg Lamp. The following Zeiss filter combi-nations were used: for rhodamine, set no. 14 (intensive); forfluorescein, set no. 17 (selective); and for coumarin, exciterfilter G 365 (ultraviolet) and as barrier filter the 'intensive'FITC exciter filter BP 450-490. For observation and pho-tography a Zeiss Neofluar (63 x/1-25) antiflex lens, thattransmits in the ultraviolet region of the spectrum, wasemployed. Micrographs were recorded on black and whiteAgfapan professional 400 negative film or Kodak Ektakrome400 slide film with the following DIN settings on theexposure meter: rhodamine, 36; fluorescein, 27-30; cou-marin, 21-24.

22 J. V. Small et al.

Results and discussion

As indicated in Materials and methods, a number ofdyes exhibiting emission in the blue region of thespectrum were investigated for their suitability asphalloidin fluorophores. Although all of them werecomplexed successfully with phalloidin, very few thenretained their capacity to bind to actin, as judged bypositive labelling of aldehyde-fixed cells or unfixedstriated muscle myofibrils in the fluorescence micro-scope. Of the three amino-selective coumarin deriva-tives tried, only one (CPITC) labelled cells stronglyenough to make prolonged observation and photogra-phy feasible. Measurements of binding affinity showedthis phalloidin analogue to bind 125 times less stronglythan unconjugated phalloidin (Fig. 1).

The absorption and emission characteristics of cou-marin-phalloidin are shown in Fig. 2. For practicalpurposes the two spectra fit closely enough to theabsorption spectra of the Zeiss u.v. excitation (365 nm)and fluorescein wide band (450-490 nm) excitationfilters for these two filters to be used, respectively, asthe excitation and barrier filters in the coumarinchannel. Using the standard u.v. filter set (no. 02) fromZeiss, single immunofluorescence images are readilyobtainable. However, overlap then occurs between thecoumarin and fluorescein channels. This can only beavoided with the above filter combination for coumarinand a narrow band excitation filter for fluorescein (seealso Materials and methods). Under these conditions,successful photographic recording was achieved withexposure times that were comparable with those usedwith rhodamine-labelled phalloidin, in the range of5-15 s (oil immersion, 63X objective). An example of ahuman skin fibroblast triple-labelled for actin (cou-marin-phalloidin) vimentin (fluorescein) and tubulin(rhodamine) is shown in Fig. 3. The lack of crossoverinto the coumarin channel is evident. In those caseswhere a very dense vimentin coil dominated one part ofthe cell we did, however, note a depletion of actin stainin the same region. We attribute this to absorption ofthe coumarin fluorescence by the neighbouring, excess

300 400 500Wavelength (nm)

600

Fig. 2. Absorption spectrum ( ) and fluorescencespectrum (excitation 387 nm; ) of coumarin-phalloidin, in water.

Fig. 3. Triple immunofluorescence combination of humanskin fibroblast stained with: A, coumarin-phalloidin;B, anti-tubulin-rhodamine; C, anti-vimentin-fluoresccin.Bar, lOjum.

Coumarin-phalloidin: a new actin probe 23

fluorescein fluophore; in other regions this'effect wasnot seen.

Three anti-bleach agents were tested for their suit-ability with the triple fluorophore combination: n-propyl gallate (Giloh & Sedat, 1982), 1,4-diazobicyclo-(2,2,2)-octane (DABCO) and phenylenediamine(Johnson et al. 1982), using Gelvatol as mountingmedium. Although DABCO (at lOOmgmP1) had amarked inhibitory effect with fluorescein and rhoda-mine, it did not noticeably reduce the bleaching ofcoumarin. Reduction of fading was obtained with n-propyl gallate (2-5mgml~l) and phenylenediamine( l rngmP 1 ) , permitting typical exposure times forrecording an image on 400 ASA/27 DIN film of 7-15 s.For coumarin it was, in addition, found important tokeep the pH around neutrality (pH 6-5 to 7-5) since atmore alkaline values (pH 8-0) an increase in back-ground fluorescence in blue as well as a reducedstaining intensity was observed.

In conclusion, the phalloidin derivative describedhere should prove generally useful for studies in whichthe distribution of actin relative to two other com-ponents in the cell may provide extra details of cyto-skeletal interactions. This has already proved to be thecase in our own recent studies on cell locomotion(Small & Rinnerthaler, 1985; Rinnerthaler, Small &Geiger, unpublished). Owing to its small size, phal-loidin readily penetrates into densely packed actinnetworks and because of its additional stabilizing actionon actin filaments it constitutes an ideal probe for thethin filament system. Hitherto, only thiol-reactivecoumarin derivatives have been applied in histo-chemical studies (Namihisa et al. 1980; Sippel, 1981).Since the present dye (CPITC) used for phalloidin isan isothiocyanate it should be equally suitable as anantibody probe and thus of more general use inimmunocytochemistry.

We thank Ms M. Hattenberger and Mrs H. Kunka forvaluable technical assistance, and Ms K. Koppelstatter forphotography.

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(Received 14 September 1987 -Accepted 2 October 1987)

24 J. V. Small et al.