Biosensors & Bioelectronics 16 (2001) 31–36

The electric charge of pigment granules in pigment cells

Martin F. Testorf a,*, Ingemar Lundstrom b, P. A, ke O8 berg a

a Department of Biomedical Engineering, Uni6ersity Hospital, Linkoping Uni6ersity, SE-581 85 Linkoping, Swedenb Di6ision of Applied Physics, Linkoping Uni6ersity, Linkoping, Sweden

Received 21 January 2000; received in revised form 20 September 2000; accepted 26 October 2000

Abstract

Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied aspromising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to findoptimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro byelectrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) weremeasured. The charge was found to be −1.5·10−16 and −1.7·10−16 C, aggregated and dispersed melanosomes, respectively,without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed. © 2001Elsevier Science B.V. All rights reserved.

Keywords: Biosensor; Electrophoresis; Melanophores; Melanosomes; Zeta potential

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1. Introduction

Highly specialised pigment cells are responsible forcommunication and camouflage in many low verte-brates, e.g. fishes and frogs. The black and dark brownpigment cell, the melanophore, responds to a specificstimulus by a rapid and highly visible phenomenon —it reorganises intracellular pigment granules to switchbetween a fully dispersed to a completely aggregatedstate, which can be observed as a colour change orfrom a dark to a brighter impression. These types ofcells are studied by many research groups for two mainreasons. Melanophores are excellent models for thestudy of cellular events like G-protein coupled recep-tors, signalling pathways, and organelle transportmechanisms. They are also very attractive as biosensors(Svensson et al., 1993; Danosky and McFadden, 1997),mainly due to their large size (diameter, :0.1 mm) andhigh optical contrast, i.e. their rapid and clearly visibleresponse to a specific receptor activation. By transfec-

tion, the specificity of the cell can be changed to matcha desired measurand. Transfection is a common biolog-ical method to introduce new DNA to make the cell,e.g. express new kinds of receptors (McClintock andLerner, 1997).

The melanophore is a large, flat cell with numerousmelanin containing pigment granules, so calledmelanosomes. Each melanosome is attached to thecytoskeleton (microtubules and actin filaments) by mo-tor proteins. When activated, the motor proteins movethe melanosomes along the cytoskeleton in an ATPconsuming process (Schnitzer and Block, 1997). De-pending on the type of stimuli, the melanosomes assem-ble in the cell centre (aggregation) or redistribute to fillthe whole cell (dispersion). See Fig. 1. Themelanosomes are dense particles, round or ellipsoidal,with a diameter of approximately 700 nm. They consistof a lipid bilayer membrane enclosing a dense core ofmelanin (McClintock et al., 1993), which is an irregular,light-absorbing polymer with several electron donorsand cation chelator properties of deprotonated hy-droxyl groups (Riley, 1997). The charge ofmelanosomes may play a role in the intracellular trans-port system.

* Corresponding author. Tel.: +46-13-222474; fax: +46-13-101902.

E-mail address: marte@imt.liu.se (M.F. Testorf).

0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 9 5 6 -5663 (00 )00130 -5

M.F. Testorf et al. / Biosensors & Bioelectronics 16 (2001) 31–3632

Fig. 1. Time sequence of an aggregating melanophore. The cell shape (outlined) is unchanged, while intracellular pigment is redistributed.

Earlier pigment cell based biosensors have utilisedoptical principles; absorption of light penetrating thecells during aggregation has been used as the measur-and. Svensson et al. (1993) studied the absorption of afish scale during aggregation induced by the pertussistoxin (Svensson et al., 1993). O8 dman et al. (1992) havestudied the velocities of melanosomes using quasielasticscattering of laser light as well as transmission changesin subcellular spots (O8 dman et al., 1992).

The aim of the present study was to determine theelectric charge of melanosomes from Xenopus lae6is(African leaf frog).

2. Materials and methods

2.1. Melanosome preparation

Melanophores from the African leaf frog, X. lae6is,were isolated and propagated in culture as describedpreviously by Daniolos et al. (Daniolos et al., 1990) andMcClintock and Lerner (McClintock and Lerner,1997). Aggregated and dispersed melanosomes wereextracted separately following the protocol of Rogers etal. (Rogers et al., 1998). Aggregation and dispersionduring 1 h was stimulated with 5 nM melatonin and 1mM a-melanocyte-stimulating hormone (a-MSH), re-spectively. The cells were lysed in 0.7× phosphate-buffered saline (PBS), pH 7.3, by homogenisation witha 273/4 gauge 1 ml syringe (three refills per ml celllysate) and the lysate was centrifuged at 600×g for 5min to separate melanosomes from unbroken cells andlarge cell fragments. The buffer also contained proteaseinhibitors (Chymostatin, Leupeptin, Pepstatin A, allsupplied by Sigma-Aldrich Inc., Stockholm, Sweden).Finally, centrifugation at 4000×g for 15 min through alayer of 80% percoll, a high density substance, sepa-rated the melanosomes from other cellular components,since melanosomes are the only cellular componentsthat are dense enough to penetrate 80% percoll. Themelanosomes were stored frozen into distilled water at−70°C, until used.

2.2. Electrophoresis chamber

The electrophoresis chamber is shown in Fig. 2. Anopen box was manufactured from glass (bottom) andperspex (walls) measuring 4×10×3 mm (width,length, depth). The two long opposite walls were cov-ered with copper electrodes connected to a voltagesource. A voltage of 1.5 V gives an electric fieldstrength of 375 V/m and a current density of less than3 mA/cm2. At these settings electrolysis at the electrodesurfaces was avoided. Melanosomes were suspended indistilled water and 0.1 ml of this suspension was in-jected in to the chamber before each electrophoresismeasurement.

2.3. Video microscopy

The movements of melanosomes were observed witha 32× objective in a microscope (Zeiss Axiovert 100M,Germany). Image sequences with 1 s image intervalwere recorded with a chilled, black and white CCDcamera (Hamamatsu C5985, Japan) and stored in a

Fig. 2. The electrophoresis chamber. Copper electrodes cover thesides (dark shaded). Particle velocity parallel to the electric field ismeasured at the centre of the chamber at different depths (dottedvertical line), and a schematic velocity profile is indicated as the solidcurve.

M.F. Testorf et al. / Biosensors & Bioelectronics 16 (2001) 31–36 33

Fig. 3. Schematic velocity profile in the electrophoresis chamber.Observed velocity 6obs, the constant electrophoretic velocity 6e, andbulk flow 6bulk due to electro-osmosis and counter flow thereof.Vertical shaded line indicates zero velocity when bulk flow and 6e areequal. Subtracting 6e from 6obs equals 6bulk.

When the electric field is applied, the forces in thechamber are immediately balanced and the particlevelocity constant. Therefore, Eqs. (1) and (2) can becombined and the effective charge of melanosomes iscalculated from its electrophoretic velocity, 6e, by solv-ing for q.

q=6 p h r 6e/E (3)

The following values have been used in the calcula-tions; h=1040·10−6 Ns/m2; r=350 nm; E=375 V/m.

To calculate q, 6e must be separated from 6obs (Fig.3). Since 6e is constant over the profile, and by assum-ing that the bulk flow is equal in both directions(average zero), averaging the whole velocity profilegives 6e.

2.5. Electrophoresis measurement

Before each experiment, the chamber was washedwith distilled water. The chamber was filled with 0.1 mlof distilled water with monodispersed melanosomes.The electrodes were short-circuited until a few secondsbefore each experiment to avoid possible initial voltagebetween the electrodes. When the voltage is applied, theforces acting on a melanosome are balanced and itsvelocity is constant. The velocity profile in the centre ofthe chamber was then recorded. This was accomplishedby, during the recording of the image sequence, succes-sively focusing on 16 different levels between the bot-tom and surface and only measuring the velocities ofmelanosomes in focus. See Fig. 2. The recordings werestored in the computer and the melanosome velocitieswere automatically computed by the software, steppingthrough the image sequence and manually marking thepositions of each melanosome. Velocity on each levelwas computed as the average velocity of threemelanosomes and this was plotted as a velocity profile.All the measurements were completed within 3 min. Inthis experiment, ten different batches from aggregatedand dispersed melanophores were measured separately.The average charge of the melanosomes of each batchwas measured as the average of nine repeated profilemeasurements.

Control experiments with polysterene particles (man-ufactured by Polysciences, Inc., USA. Diameter1.07290.019 micrometer) were made. After thepolystyrene particles had been diluted (nine dilutions)in distilled water, charge measurements were carriedout as with the melanosomes. The measurement strat-egy is summarised in Fig. 4.

3. Results

The results of the electrophoretic velocity measure-ments are shown in Table 1 and the electric charge in

computer (PC with frame grabber IC-PCI, ImagingTechnology, USA). Image analysis was made with thesoftware Optimas 6.2, Media Cybernetics, LP, USA.

2.4. Theory

When the voltage is applied in between the electrodesin the electrophoresis chamber, three forces act on themelanosomes. First, the electrophoretic force, FE (N),acts on every charged particle

FE=qE (1)

where q (C) is the charge of the particle and E [V/m] isthe electric field strength. The electric field acts on theeffective charge of the melanosome, when its initialcharge to some extent is cancelled out by oppositelycharged ions in the surrounding media (the doublelayer). The effective charge is strongly dependent on pHin the solution (see e.g. Bouriat et al., 1999). The pH inthe measurements was observed to be close to neutral(6.8–7.6).

Secondly, the drag force when melanosomes move inwater (Stokes force), FS,

FS=6 p h r 6 (2)

where h (Ns/m2) is the viscosity of water, r (m) is theparticle radius (assumed to be spherical) and 6 (m/s) isthe velocity.

Thirdly, electro-osmotic forces arise at the bottom ofthe chamber and create a bulk flow (Hunter, 1981;Hiemenz and Rajagopalan, 1997). The observed veloc-ity of melanosomes is a superposition of the elec-trophoretic velocity and the bulk flow from theelectro-osmotic effects resulting in an observed velocityprofile schematically shown in Fig. 3.

M.F. Testorf et al. / Biosensors & Bioelectronics 16 (2001) 31–3634

Fig. 4. For each preparation of melanosomes (batch), nine velocityprofiles were measured with three melanosomes on each level in theprofile.

have to be considered. The charge of melanosomes is ofthe same order of magnitude as 1000 electrons and it isreasonable that the charge has chemical implicationsand maybe also physical. The magnitude of the chargeof the melanosome may serve as a source of informa-tion to quantify properties such as phosphorylation,distribution of motor proteins and mechanisms behindmelanosome movement.

We suggest the following possible origins of thecharge of melanosomes. Melanin itself is negativelycharged. Proteins bound to the membrane ofmelanosomes may have negative groups. Motorproteins associated to melanosomes can be phosphory-lated with negative phosphate groups. If it is suggestedthat melanin is the main origin of the charge of amelanosome, its charge would be proportional to itsvolume. The volume is proportional to the radius to thethird power, while electrophoretic velocity is propor-tional to charge divided by radius (melanosomes as-sumed to be spherical). This means that if charge isproportional to volume, the observed electrophoreticvelocity would be proportional to radius squared. Sizemeasurements of melanosomes show a variation inradius of 350980 nm (Testorf et al., 2000). This varia-tion, 922%, squared would foresee a variation ofabout 948% in electrophoretic velocity. Since the ob-served variation is only about 912%, the assumptionthat charge is proportional to volume seems to beincorrect. The conclusion from this is that the charge ofmelanosomes is not likely to mainly originate frommelanin. Charge uniformly distributed on the surface ofthe melanosomes would give an expected variation of922%. It appears, therefore, that the charge of themelanosomes has a rather specific origin. It can benoted that if the charge is evenly distributed on thesurface of the melanosomes, there would be a chargedensity of approximately 650 mm−2 or 40 nm betweeneach charge.

The described measurements gave several averageswith adherent variance as the result; between batches,between velocity profiles within a batch, and betweenindividual melanosomes on every level in the velocity

Table 2. The average charges of aggregated and dis-persed melanosomes were equal with no significantdifference. Since three melanosomes were measured ateach level as described in Fig. 4, 4320 melanosomeswere studied in all ten batches totally (aggregated anddispersed melanosomes, respectively). The variation incharge in percentage is expressed as the S.D. in velocitybetween melanosomes on the same level in the velocityprofile, divided by the average of a batch.

A representative example of a measured velocityprofile is shown in Fig. 5.

4. Discussion

Our study shows that melanosomes are negativelycharged and although a variation in charge betweenindividual melanosomes exist, the charge does not seemto change when the cell turns from dispersed to aggre-gated. The results suggest that to fully understand theprocesses in melanophores, interaction between charges

Table 1Electrophoretic velocitiesa

Aggregated melanosomes Dispersed melanosomes Polystyrene particles

Ten batches. A6erage electrophoretic 6elocity8.293.3 9.494.5Mean9S.D. (mm/s) 4.791.1

948 923941S.D./mean (%)

Nine profiles926 919 924Typically S.D./mean (%)

Melanosome triplesTypically S.D./mean (%) 912 911 922

a A–C refer to Fig. 4

M.F. Testorf et al. / Biosensors & Bioelectronics 16 (2001) 31–36 35

Table 2Charge of melanosomes and polystyrene particles

Aggregated melanosomes Dispersed melanosomes Polystyrene particles

−1.7Average charge (10−16 C) −1.2−1.5911Variations (%) (see Section 4) 922912

profile. Knowledge about the origin of the variancesgives an important understanding of the results. Todistinguish between variations due to methodologicalconditions and natural variations, polystyrene particleswere measured with no step involved corresponding tothe melanosome preparation. The variance betweenbatches is twice as high for melanosomes than forpolystyrene particles. This could mean that during thepreparation of melanosomes from melanophores, thecharge of melanosomes may change and/or naturalvariations between melanosomes are larger than be-tween polystyrene particles. But, variation between in-dividual particles was twice as high for polystyreneparticles than melanosomes, and there was a small S.D.between profiles in batches with the highest elec-trophoretic velocity (not shown). Altogether, this seemsto indicate that the charge is actually different fordifferent melanosome batches.

Variations between different velocity profiles fromthe same batch reveal errors in the assumption that theaverage bulk flow in each vertical plane in the chamberis zero. There is some net out-of-plane-flow and thisvaries from each measurement and results in the differ-ence between the nine repeated profile measurements ofa batch.

To further separate methodological variations andvariations occurring naturally between melanosomes, itis argued that natural variations are observed withinone level in a profile. In every such level threemelanosomes were studied and these are the only mea-surements during exactly the same methodological con-ditions (e.g. bulk flow). The velocity of melanosomeson each level in the profile was measured during 3–10s and was done very accurately with image analysis asdescribed above. The variation within a level was nor-mally 11–12% of the computed average velocity and isassumed to be the natural variation in the charge ofmelanosomes. This variation was computed as a per-centage of the average velocity of the batch, that is theaverage of the nine profiles in a batch, and not as apercentage of the average velocity in the particularlevel. This is because the variation did not correlate tothe observed velocity (electrophoretic+bulk flow ve-locity) and, therefore, was assumed to originate fromvariation in charge rather than variation in bulk flow.

No correlation was found between pH and elec-trophoretic velocity (and charge). However, a linearcorrelation between pH and charge does exist, as stated

by others (Ware, 1974; Bouriat et al., 1999). But, allmeasurements in this study had pH in the range of6.4–7.6, which was narrow enough to avoid contribu-tion to the deviation in the measurements. The naturalenvironment of melanophores has pH 7.3 and the effec-tive charge of melanosomes measured in this study is,therefore, likely to be biologically relevant.

The electro-osmosis can be explained as follows.Water on an ordinary glass surface gives a negativelycharged surface. When the surface is negativelycharged, there will be a net excess of positive ions in theadjacent liquid and as they move under influence of theapplied field they will carry the liquid with them. In thisway a flow of the liquid is induced. As the chamber isclosed a parabolic counter flow (6bulk in Fig. 3) willappear in the centre of the chamber with the samedirection as the electrophoretic force acting on themelanosomes (Wagenen et al., 1976).

Every velocity profile was measured within 3 min.During longer measurements a decrease in melanosomevelocity was observed, probably due to polarisation ofthe electrodes. Commercial devices for measurement ofparticle charge (zeta-potential) were not used in thisstudy, since they require large amounts of the sample

Fig. 5. A representative velocity profile as the average of nine profilesfrom the same melanosome batch. The error bars are 1 S.D.

M.F. Testorf et al. / Biosensors & Bioelectronics 16 (2001) 31–3636

and have limitations when measuring particles less thanone micrometer in diameter.

As a new biosensor application, taking advantage ofthe charge of melanosomes, we speculate in usingmelanophores together with electric fields to introducean electrophoretic force on melanosomes that couldcounterbalance the intracellular migration forces duringaggregation. The magnitude of the electric field neededto prevent aggregation would be a measure on theconcentration of the stimuli to be measured and de-tected. This study shows no significant difference incharge between dispersed and aggregated melanosomes.This result facilitates the above idea since the fieldstrength has to be adjusted only to the motion and notalso to a change in charge. The intracellular motorproteins exert a force on the melanosomes in the orderof 10−12 N (Hunt et al., 1994). The results from thisstudy show that an electric field of 10 V/mm is enoughto create a force big enough to compete with themotion of melanosomes during aggregation. A secondpossibility would be to study the charge distribution inthe cell as the measurand. This could probably beperformed remotely without disturbing the cell func-tion. These ideas are now being tested in ourlaboratory.

Acknowledgements

We acknowledge the receipt of the grant ‘Pigment-cellsbaserade biosensorer’ from the Swedish ResearchCouncil for Engineering Sciences. (Dnr 98–573). Themelanophores were a generous gift of Dr Michael

Lerner, Southwestern Medical Centre, University ofTexas, Dallas, USA. The melanophores were cultivatedby Annika Karlsson, Division of Pharmacology,Linkoping University, Sweden.

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