Electron microscopy as a quantitative method for investigating tau fibrillization

ANALYTICAL

Analytical Biochemistry 329 (2004) 238–246

BIOCHEMISTRY

www.elsevier.com/locate/yabio

Electron microscopy as a quantitative method for investigatingtau fibrillization

Mihaela Neculaa and Jeff Kuretb,*

a Biophysics Program, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USAb Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, Columbus, OH 43210, USA

Received 4 December 2003

Available online 30 April 2004

Abstract

Fibrillization of tau protein is a hallmark lesion in Alzheimer�s disease. To clarify the utility of electronmicroscopy as a quantitative

assay for tau fibrillization in vitro, the interaction between synthetic tau filaments and carbon/formvar-coated grids was characterized

in detail. Filament adsorption onto grids was hyperbolic when analyzed as a function of time or bulk protein concentration, with no

evidence for competitive displacement or elution from other components in the reaction mixture. Filament length measurements were

linear with filament concentration so long as the concentration of total tau protein in the sample was held constant, suggesting that

measurement of filament lengths was accurate under these conditions. Furthermore, exponential filament length distributions were not

significantly affected by adsorption time or filament concentration, suggesting that preferential binding among filaments of differing

lengths was minimal. However, monomeric tau protein was found to be a strong competitor of filament adsorption, indicating that

comparison of filament length measurements at different bulk tau concentrations should be interpreted with caution.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Tau; Amyloid; Fibrillization; Adsorption; Interfacial concentration; Microscopy

Alzheimer�s disease (AD)1 is characterized in part by

the aggregation of tau protein into neurofibrillary le-

sions [1]. Although amorphous aggregation dominates

early stages of disease [2,3], cognitive decline correlates

with the fibrillization of tau into ordered filaments

containing cross b-sheet structure [2,4]. Therefore,characterization of the fibrillization reaction is of central

importance for clarifying the underlying events in

tauopathic neurodegenerative diseases such as AD.

Quantitative assays for fibrillization reactions are

limited but include dye-based fluorescence spectroscopy

[5,6], static and dynamic light scattering [7,8], and sed-

imentation [9], each of which has specific limitations and

weaknesses [10]. Above all, none of these solution-based

* Corresponding author. Fax: 1-614-292-5379.

E-mail address: [email protected] (J. Kuret).1 Abbreviations used: AD, Alzheimer�s disease; AA, arachidonic

acid; N774, (2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiaz-

ole); EM, electron microscopy, PHF, paired helical filament; DMSO,

dimethyl sulfoxide.

0003-2697/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2004.02.023

methods involve direct visualization of fibrils, which is

critical for assessing filament morphology and length

distributions. Thus electron microscopy has emerged as

an essential adjunct to solution methods for character-

ization of tau fibrillization [11]. But the utility of the

method for rigorous quantitation has been questioned.On the one hand, experience with authentic filament

preparations suggests that filament lengths estimated by

electron microscopy are linear with filament concentra-

tion and therefore reliable [12]. On the other hand it has

been claimed on the basis of experience with synthetic

filaments prepared with heparin that quantitation of fi-

bril lengths using electron microscopy is problematic

[13,14].We employ electron microscopy as a quantitative

assay for synthetic tau filaments formed from re-

combinant tau protein and fatty acid inducer [9,11].

Fibrillization occurs in solution, as with other methods,

but resultant filaments are detected only after

adsorption of reaction products onto the hydrophobic

surface of grids coated with formvar (a polyvinyl formal

mail to: [email protected]

M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246 239

polymer) and carbon. Thus adsorption phenomena havea large influence on the accuracy of filament length

measurements by this method. Adsorption of proteins

onto various surfaces has been studied for many

proteins, including fibrinogen [15], wheat proteins [16],

serum albumin [17], lysosyme [18], insulin [19], and

b-lactoglobulin [20]. From such experiments it was

shown that adsorption depends upon temperature, pH,

ionic strength, and protein concentration [21–25]. Inaddition, adsorption from protein mixtures can be a

competitive and dynamic process, where initially ad-

sorbed proteins can undergo conformational changes

and displace other proteins in a time- and concentra-

tion-dependent manner [15,26,27]. The later process is

known as the Vroman effect, and it can produce non-

linearity in adsorption-based assays [28,29].

Here we characterize the interaction of tau fibrilliza-tion reaction products with formvar/carbon-coated grids

as a function of protein concentration and adsorption

time. In addition, we investigate the effect of soluble tau

protein, fatty acid, and tau fibrillization inhibitor N744

on fibril–grid surface interaction. Results show the

conditions under which electron microscopy can be used

to give accurate estimates of tau filament length.

Materials and methods

Materials

Recombinant double mutant htau40C291A;C322A and

htau37 were expressed and purified as described previ-

ously [8,30,31]. AA (Fluka, Milwaukee, WI) was dis-solved in 100% ethanol and stored under argon gas at

)80 �C until used. Tau polymerization inhibitor N744

was the generous gift of Neuronautics (Evanston, IL)

and was dissolved and stored at )20 �C in DMSO.

Formvar/carbon-coated grids (300-mesh), glutaralde-

hyde, and uranyl acetate were from Electron Micros-

copy Sciences (Ft. Washington, PA). Carboxylate-

conjugated polystyrene microspheres (90 nm diameter;molecular area¼ 12�A2/eq) were obtained from Bangs

Laboratories (Fishers, IN).

Tau fibrillization

Tau isoforms htau40C291A;C322A or htau37 (4–10 lM)

were incubated without agitation in Assembly Buffer

(10mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonicacid, pH 7.4, 100mM NaCl, and 5mM dithiothreitol)

at 37 �C for up to 24 h in the presence of either AA

(0–200 lM) or carboxylate-substituted polystyrene

microspheres (124 pM). Reactions were terminated with

glutaraldehyde treatment (2% final concentration) for

5–30min, diluted 0- to 40-fold in Dilution Buffer

(Assembly Buffer containing 2% glutaraldehyde) alone

or containing various additions, and subjected toelectron microscopy assay as described below. When

present, tau fibrillization inhibitor N744 was accompa-

nied by DMSO vehicle, which was limited to 1% in all

reactions.

Transmission electron microscopy

Aliquots (50 ll) of fixed reactions were adsorbed(0.25–15min) onto 300-mesh formvar/carbon-coated

copper grids. The resultant grids were washed with

water, stained (1min) with 2% uranyl acetate, washed

again with water, blotted dry, and viewed in a Phillips

CM 12 microscope operated at 65 kV. Random images

from each experimental condition were captured on film

at 8000- to 22,000-fold magnification, digitized, cali-

brated, and imported into Optimas 6.5.1 for quantita-tion of filament length and number as described

previously [30]. Interfacial filament concentration (Cf ) is

defined as the summed lengths of all filaments >50 nm in

length per unit area and is reported in units of nm/

lm2 � SD as calculated from three to six random images

per condition. Interfacial microsphere concentration

(CM) is defined as the number of microspheres per unit

area and is reported in units of microspheres/lm2 � SDas calculated from three to six random images per

condition.

Analytical methods

Hyperbolic adsorption data were fit to the Toth iso-

therm [32],

C ¼ Cmax

ax

ð1þ ðaxÞbÞ1=b;

where C is the interfacial concentration measured at

bulk concentration x, Cmax is the interfacial concentra-

tion at saturation, a is a constant, and b is the Toth

coefficient reported� SE without units.

Filament length distributions were fit to the expo-

nential equation,

y ¼ aebx;

where y is the percentage of all filaments filling a bin of

length interval x and b is a constant reported in units oflength�1 � SE.

Results of linear regression analysis are reported�SE.

Results

Effect of dilution on filament adsorption

Experimentation began with mutant tau con-

struct htau40C291A;C322A because it simplifies and speeds

240 M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246

filament quantitation [8]. Adsorption of filamentoushtau40C291A;C322A onto formvar/carbon-coated EM grids

was examined as a function of bulk tau concentration

after serial dilution of glutaraldehyde-fixed fibrillization

products into Dilution Buffer at room temperature.

Adsorption times of 1 and 15min were studied. Resul-

tant plots were linear up to 1 lM total tau concentration

for 1-min adsorption and up to 0.5 lM tau for 15-min

adsorption, with initial slopes dependent on adsorptiontime (Fig. 1). Above these tau concentrations, isotherms

abruptly became nonlinear and appeared to approach

saturation. There was no indication of decreasing fila-

ment adsorption with decreasing dilution, suggesting

that adsorbed fibrils were not displaced or eluted by

other components of the assembly system such as mo-

nomeric tau or AA (i.e., there was no Vroman effect).

Extended binding linearity followed by abrupt satu-ration is characteristic of type C2 adsorption behavior in

the empirical Giles et al. [33] classification system.

Therefore, the Toth isotherm was used to fit these data

because it accurately models linearity at low sorbate

concentrations [34]. Saturation as estimated by extrap-

olation from the resultant isotherms was 3150� 80

(Toth coefficient¼ 4.1� 1.1) and 3510� 40 (Toth coef-

ficient¼ 4.5� 1.2) nm/lm2 length for 1- and 15-minadsorption times, respectively. Assuming a mass per unit

length of 75� 17 kDa/nm [35] yielded molecular areas of

15–21 cm2/lg filamentous tau protein at saturation.

These values are below typical values determined for

globular proteins in single-component experiments [36].

Fig. 1. Tau filament adsorption isotherm. Filaments prepared (3 h at

37 �C) from recombinant double mutant htau40C291A;C322A (4lM) in

the presence of AA inducer (75lM) were fixed in 2% glutaraldehyde,

serially diluted in Dilution Buffer, adsorbed onto carbon/formvar-

coated grids for either 1min (j) or 15min (�), and then viewed by

transmission electron microscopy after negative staining. Each point

represents Cf (the interfacial concentration of filaments >50 nm in

length) as a function of dilution (where a dilution of unity, 4lM total

tau), whereas each curve represents the best fit of data points to the

Toth isotherm. Under these conditions, adsorption increased linearly

with concentration until an abrupt break point (type C2 behavior in

the nomenclature of Giles et al. [33]).

These data suggest that filament adsorption ontocarbon-coated grids is linear only up to a threshold

concentration related to saturation on the grid surface

and that filament length measurements made above this

level are not proportional to filament concentrations in

solution.

Adsorption timecourse

To investigate the kinetics of filament adsorption,

htau40C291A;C322A filaments were prepared from 4 lMtotal tau and adsorbed onto grids neat or diluted 5-fold

in Dilution Buffer. Neat reaction product adsorbed and

saturated within 15 s, with interfacial filament concen-

tration remaining constant over the entire 15-min time

course (Fig. 2). Diluted reaction products adsorbed

more slowly with hyperbolic kinetics (Fig. 2). Bothcurves approached saturation monotonically without

evidence of transient adsorption (a manifestation of the

Vroman effect). These data suggest that adsorption time

is an important variable in measurement of filament

lengths and therefore samples investigated by EM

should be compared only if they were adsorbed onto

grids for identical periods of time.

Interfering substances

In addition to filaments, fibrillization reactions con-

tain fatty acid and unincorporated tau protein, each of

which could potentially compete with filament binding

and contribute to molecular crowding at the grid

Fig. 2. Kinetics of tau filament adsorption onto grids. Filaments

prepared (3 h at 37 �C) from recombinant double mutant

htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) were

fixed in 2% glutaraldehyde and serially diluted in Dilution Buffer. The

adsorption of undiluted fibrils (�) and 5-fold-diluted fibrils (j) onto

carbon/formvar-coated grids was then measured as a function of ad-

sorption time at room temperature. Each point represents Cf as a

function of adsorption time, whereas each line represents the best fit of

the data points to a rectangular hyperbola. Although undiluted reac-

tion products adsorbed within 15 s, no time-dependent decrease in Cf

was observed under these conditions.


surface. In fact these may account for the relatively highmolecular areas associated with filament saturation.

Moreover, both proteins and anionic surfactants can

elute adsorbed proteins when present at high concen-

trations and therefore complicate quantitation [37]. To

determine which components of the reaction mixture

contributed to crowding, filaments synthesized from

4 lM htau40C291A;C322A were diluted 5- to 10-fold in

Dilution Buffer containing various concentrations ofAA or monomeric tau and analyzed by electron mi-

croscopy after 1- or 15-min adsorption times. Results

showed that AA concentrations up to at least 50 lM did

not compete for filament adsorption regardless of dilu-

tion or incubation time (Fig. 3A). In fact, concentra-

tions up to 200 lM appeared to be without effect,

although samples adsorbed 1min containing >100 lMAA or adsorbed 15min containing >50 lM AA werenot quantified due to the fibril clumping that occurred in

this assay format. These data suggest that normal

working concentrations of anionic surfactant inducers

Fig. 3. Anionic surfactants do not interfere with filament adsorption.

Filaments prepared (3 h at 37 �C) from recombinant double mutant

htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) were

fixed in 2% glutaraldehyde and diluted into Dilution Buffer containing

various concentrations of AA or small-molecule tau fibrillization in-

hibitor N744. Each point represents interfacial filament concentration

(Cf ) as a percentage of control adsorption conducted in the absence of

additions, whereas each line represents linear regression analysis of the

data points. (A) Filaments were either diluted 5-fold and adsorbed

1min (j) or diluted 10-fold and adsorbed 15min (�) in the presence of

varying AA concentrations. AA did not interfere with adsorption at

concentrations up to 100lM (although these concentrations led to

filament clumping in the 15-min format, thereby making quantitation

questionable). (B) Filaments were either diluted 5-fold and adsorbed

1min (d) or diluted 10-fold and adsorbed 15min (s) in the presence of

varying concentrations of N744. Under these diverse conditions, N744

at concentrations up to 50 lM did not interfere with filament ad-

sorption.

(0–100 lM) do not interfere with assay of tau fibrilliza-tion.

The ability of small-molecule agents to interfere with

adsorption also was examined using tau fibrillization

inhibitor N744, a member of the Congo red family of

dyes [38]. These compounds are thought to bind all

along the length of filaments containing cross b-sheetconformation [39]. Dilution of filaments into Dilution

Buffer containing up to 50 lM N744 did not affectfilament adsorption (Fig. 3B), suggesting that the

presence of small-molecule ligands such as N744 at

working concentrations (0–4 lM) does not affect assay

performance.

In contrast, the presence of monomeric tau protein in

Dilution Buffer was strongly competitive above 1 lM for

1-min adsorption and 0.4 lM for 15-min adsorption

(Fig. 4). These data suggest that bulk tau protein maycontribute to the saturation of interfacial filament con-

centration observed as a function of total tau concen-

tration and that measurement of tau fibrillization at

different tau concentrations can misstate true levels of

fibrillization.

Together these data indicate that fibrillization in the

presence of varying inducer and inhibitor concentrations

can be directly compared at constant tau concentration.However, measurements made in the presence of vary-

ing tau concentrations cannot be directly compared

unless diluted to linearity.

Fig. 4. Monomeric tau protein interferes with filament adsorption.

Filaments prepared (3 h at 37 �C) from recombinant double mutant

htau40C291A;C322A (4lM) in the presence of AA inducer (75lM) and

fixed in 2% glutaraldehyde were diluted 5-fold into Dilution Buffer

containing various concentrations of monomeric htau40C291A;C322A,

adsorbed onto carbon/formvar-coated grids for either 1min (j) or

15min (�), and then viewed by transmission electron microscopy after

negative staining. Each point represents interfacial filament concen-

tration (Cf ) as a percentage of control adsorption conducted in the

absence of additions, whereas each line is drawn to aid visualization

without being fit to a model. Monomeric tau protein competes with

filament adsorption at concentrations above 1lM in the 1-min and

0.4 lM tau in the 15-min adsorption paradigms.


Assay linearity at constant total protein concentration

The above data suggest that, despite the complexity

of reaction mixtures, adsorption behavior reduces to a

binary system (monomeric and filamentous tau popu-

lations) at constant temperature and buffer conditions.

These two components can directly compete for ad-

sorption, but the lack of visible Vroman effect in both

time course and dilution formats suggest that exchangeand elution reactions are minimal. Under these condi-

tions, adsorption of each component of a binary system

should reflect their relative bulk concentrations in so-

lution [15,40]. Therefore the interfacial binding of

filaments should be a linear function of filament con-

centration so long as the competing agent, monomeric

tau protein, is held constant and saturation is not

reached. To test this hypothesis, aliquots of filamentswere diluted up to 40-fold in Dilution Buffer containing

varying concentrations of tau such that the final total

tau concentration remained constant. Diluted samples

were then adsorbed onto grids (1-min incubation time)

and examined by transmission electron microscopy.

Two tau isoforms were used: htau40C291A;C322A at 4 lMbecause this four-repeat isoform fibrillizes efficiently at

this concentration and htau37 at 10 lM because three-repeat isoforms nucleate less efficiently and require

higher concentrations for reliable filament length mea-

surements [30]. The resultant plots of filament interfacial

concentration versus dilution were linear for both tau

isoforms (Fig. 5). Linearity was observed after 15-min

adsorption times also (data not shown). These data

Fig. 5. Filament adsorption is linear at constant total protein con-

centrations. Filaments prepared from 4 lM htau40C291A;C322A (3 h at

37 �C; 75 lM AA) or 10lM htau37 (24 h at 37 �C) in the presence of

AA inducer were fixed in 2% glutaraldehyde and then serially diluted

into Dilution Buffer containing various concentrations of monomeric

tau protein so that the final concentration of tau was held constant at

either 4lM (j, htau40C291A;C322A) or 10 lM (�, htau37). Each point

represents Cf as a function of dilution after 1-min adsorption, whereas

each line represents linear regression analysis of the data points. Fil-

ament adsorption is linear with filament concentration at constant

total tau concentration.

confirm linearity of filament length measurements in thepresence of constant total tau concentration and suggest

that length measurements are reliable below saturation

so long as this condition is met.

Filament length distributions

Although no displacement of filaments as a function

of time or tau concentration was observed, it is possiblethat filaments of differing lengths adsorb at different

rates, resulting in biased apparent length distributions.

To address this issue, the length distribution of filaments

prepared from 4 lM htau40C291A;C322A and 75 lM AA

was determined as a function of adsorption time and

dilution at constant total tau concentration. All length

distributions were exponential, consistent with the time-

dependent fibrillization pathway induced by AA (Fig. 6)and therefore were quantified using exponential slope

parameter b as described under Materials and methods.

Undiluted samples adsorbed for 1 or 15min had bvalues of )5.34� 0.67 and )5.02� 0.64 lm�1 (Fig. 6),

consistent with previously published results (b ¼ �5:4�0:2 lm�1 at 3 h; Fig. 1 in [9]). Similarly, the length dis-

tributions of samples diluted 4-fold and adsorbed for 1

or 15min had b values of )5.55� 0.71 and)5.83� 0.70 lm�1, although the number of filaments

adsorbed onto grids was 3- to 5-fold lower in these di-

luted samples (Fig. 6). These data, together with data

presented in Fig. 5, suggest that length distributions are

Fig. 6. Length distributions are independent of tau protein concen-

tration and adsorption time. Filaments formed from htau40C291A;C322A

incubated (3 h at 37 �C) in the presence of 75lM AA were fixed in 2%

glutaraldehyde and either diluted 4-fold into Dilution Buffer (j;�) or

used undiluted (d;s). All filament populations were then adsorbed

onto grids for either 1min (j;d) or 15min (�;s). The lengths of

filaments P 50 nm in length were then measured from digitized images

and plotted. Each data point represents the number of analyzed fila-

ments segregating into consecutive length intervals (100-nm bins),

whereas each line represents the best fit of data points to an expo-

nential regression. Length distributions remained exponential with

nearly identical slopes regardless of protein concentration or adsorp-

tion time.


not dependent on adsorption time when assayed atconstant total tau concentration.

Microsphere adsorption is tau protein dependent

Anionic microspheres are an important new class of

tau fibrillization inducer. Although their mechanism of

induction closely resembles that of AA, their adsorption

properties may differ substantially. To clarify this issue,adsorption of 124 pM anionic microspheres (90 nm di-

ameter; 12�A2/eq molecular area) onto grids was deter-

mined after 24-h incubation in the presence or absence

of 4 lM htau40C291A;C322A. In the absence of tau protein,

plots of interfacial microsphere concentration (CM) were

linear with dilution (Fig. 7A). This pattern, termed type

Fig. 7. Anionic microsphere adsorption isotherms. Anionic micro-

spheres (124 pM) were incubated (24 h at 37 �C) with (j) or without

(�) 4 lM htau40C291A;C322A under fibrillization conditions and then

treated with 2% glutaraldehyde, serially diluted in Dilution Buffer, and

adsorbed onto grids for 1min. The interfacial concentration of micr-

ospheres (CM; j;�) and tau filaments (Cf ; s) were then estimated

from electron micrographs. (A) Each point represents C measured as a

function of dilution (where a dilution of unity¼ 124pM microspheres).

Although microsphere adsorption was linear with dilution in the ab-

sence of tau (�; line represents linear regression), it was hyperbolic in

the presence of tau (j; line represents best fit to the Toth isotherm),

suggesting that microsphere adsorption was protein concentration

dependent. Under these conditions, Cf also varied hyperbolically with

bulk tau concentration (s; line represents best fit to the Toth iso-

therm). (B) Replot of CM versus Cf determined at different microsphere

concentrations (error bars were omitted for clarity). Because filaments

remained associated with microspheres after nucleation, Cf varied

linearly with CM.

C1 in the nomenclature of Giles, is typical of adsorbantswhen present well below saturation [41]. In contrast,

plots of interfacial microsphere concentration against

dilution were hyperbolic in the presence of tau (Fig. 7A).

Fitting the data to the Toth isotherm yielded a Toth

coefficient of 1.3� 0.4 and a saturating interfacial con-

centration of 1.28� 0.12microspheres/lm2. Toth coef-

ficients of �1 reduce to the Langmuir isotherm [32], as

reflected in the classic rectangular hyperbolic shape ofthe curve (Giles type L2). These data show that tau

protein influences microsphere adsorption behavior and

that assay of tau fibrillization using microspheres in-

stead of AA as inducer does not alleviate the nonlin-

earity produced by the different concentrations of tau

protein. Nonetheless, because filaments grow from in-

ducer surfaces and remain associated after nucleation

[9], microspheres could potentially serve as easily mea-sured internal standards for adsorption. To test this

hypothesis, the study was extended to include mea-

surement of interfacial filament concentration (Cf ). Re-

sults showed that Cf paralleled CM as a function of

microsphere dilution (Fig. 7A) and that plots of Cf

versus CM were linear at all dilutions examined

(Fig. 7B). These data confirmed that the Cf=CM ratio

was constant even when microsphere adsorption wassaturating and suggested that microspheres could be

used to normalize electron-microscopy-based length

measurements performed under nonlinear adsorption

conditions.

Discussion

Electron microscopy-based assays for tau fibrilliza-

tion differ from solution-based methods by employing

an adsorption step onto hydrophobic grids prior to

quantitation of reaction products. The data presented

here suggest that filament adsorption is linearly related

to concentration in solution up to saturation when

measured at constant pH, ionic strength, adsorption

time, and AA inducer and tau concentrations. This be-havior is maintained in the presence of varying con-

centrations of AA inducer, suggesting that filament

lengths can be assayed by electron microscopy when this

parameter is varied. Moreover, filament adsorption is

not modulated by Congo red-related dyes such as N744

up to 50 lM concentrations.

The major variable in assay performance appears to

be bulk tau concentration, which competes with fila-mentous tau adsorption on grids. Therefore, we propose

that tau adsorption phenomena reduce to a binary sys-

tem consisting of monomeric and fibrillized tau popu-

lations when examined at constant pH, ionic strength,

and adsorption time. The ratio of interfacial concen-

trations for two competing adsorbates (termed 1 and 2)

has been modeled by the expression [15]

� � � �244 M. Necula, J. Kuret / Analytical Biochemistry 329 (2004) 238–246

C2

C01

¼ 1þ kd1k01

ka2ka1

C2

C1

þ 1þ ka2ka1

C2

C1

kexk01

C2; ð1Þ

where the ratio of interfacial concentrations (C) is a

function of each adsorbate�s bulk concentration (C) and

relative rates of absorption (ka), desorption (kd), ex-

change (kex), and transformation to an irreversibly ad-sorbed state (k01 in the case of the first adsorbate). The

absence of Vroman effects accompanying tau filament

adsorption suggests that exchange reactions for fila-

mentous tau are minimal over the 15-min time courses

investigated here (i.e., kex � 1). Furthermore, adsorp-

tion of filaments appears to be tenacious, suggesting that

rates of desorption also are minimal (i.e., kd � 1). If the

exchange and desorption rates of monomeric tau alsoare low, then the ratio of interfacial concentrations re-

duces to the ratio of bulk concentrations of each com-

ponent [15,40]. In other words, adsorption isotherms for

tau filaments reflect not only filament bulk concentra-

tion in solution but also the bulk concentration of

nonfibrillar tau. As a result, estimates of Cf differ when

constant amounts of tau filaments are adsorbed in the

presence of varying concentrations of total tau protein.These considerations extend to filaments of different

lengths also. Because the adsorption of higher-molecu-

lar-weight polymers is thermodynamically preferred

over the lower-molecular-weight fibrils, they would be

expected to exchange with shorter polymers over time

[42]. But kinetic curves show no signs of such behavior

(displacement), again presumably due to minimal de-

sorption and exchange reactions [43,44]. In any event,length distributions do not vary as a function of ad-

sorption time or concentration, suggesting that such

measurements are reliable when performed at constant

total tau concentration.

Published data are consistent with this model. First,

authentic filaments diluted in the absence of competing

substances have been shown to adsorb linearly with fil-

ament concentration [12]. This isotherm, termed C1 inthe Giles system [33], is typically observed at sorbate

concentrations far below saturation [41]. Thus, linearity

depends on the initial concentration of purified tau fil-

aments in the preparation. Second, filaments prepared in

vitro from synthetic inducers such as fatty acids adsorb

linearly as part of a multicomponent system so long as

the tau concentration is held constant. Thus measure-

ments of inducer efficacy at constant tau protein, forexample, are directly comparable [9]. Third, synthetic

tau filament adsorption is nonlinear when performed at

widely different total tau concentrations, which have

been used in studies characterizing tau fibrillization in

the presence of heparin [14]. Moreover, heparin-induced

reactions are typically conducted at 10–100 lM total tau

protein, far above the concentrations used here [6,14].

Although Vroman effects are not apparent up to 10 lMtau (the highest concentration used in our experiments),

use of higher concentrations may drive exchange reac-tions and further complicate quantitation of filament

lengths.

The nature of fibril–surface interaction may explain

the lack of the Vroman effect at low (610 lM) tau

concentrations. Although driving forces for adsorption

of proteins include hydrophobic and electrostatic inter-

actions [45], binding to the hydrophobic surface of

formvar/carbon-coated grids is presumably dominatedby hydrophobic interactions. Strong hydrophobic in-

teractions between the fibrils and the surface probably

contribute to tenacious fibril adsorption and the lack of

fibril displacement [29], which may further increase due

to glutaraldehyde treatment. Indeed, the Vroman effect

diminishes with increasing surface hydrophobicity

[15,29,46]. Tenacious adsorption to surfaces also is

promoted by entropy gains resulting from protein con-formational changes subsequent to adsorption [25].

However, changes in monomeric tau or tau filament

conformation on the grid surface are unlikely under

conditions used herein because samples were fixed with

glutaraldehyde prior to adsorption and because mono-

meric tau is natively unfolded [47].

Although the above considerations adequately de-

scribe the macroscopic behavior of tau adsorption,events at the molecular level are unclear. Indeed, the

molecular basis for continuous-shape adsorption iso-

therms is not understood for purified analytes let alone

the complex mixture described here [41]. But the Giles

type C2 isotherm and high Toth coefficient accompa-

nying dilution of tau fibrillization products are consis-

tent with secondary events accompanying adsorption so

that the effective surface area appears to increase [32,41].This behavior may be unique to surfactants such as AA

because anionic microspheres, a chemically distinct class

of fibrillization inducer, do not behave in this way, in-

stead adopting the classic Langmuir adsorption iso-

therm when diluted in the presence of tau protein.

In summary, accurate quantitation of filament length

distributions and total filament length can be performed

under conditions of constant bulk tau concentration.Interference from anionic surfactant (6200 lM) and

fluorescent dyes (650 lM) are minimal under these

conditions. Filament lengths can also be determined at

different total tau concentrations, but samples must be

diluted so that the interfacial tau filament concentration

is not saturating. Even then, care must be taken so that

competition from nonfibrillar tau is minimal. We pro-

pose two solutions to this problem. First, samples can bediluted into solutions containing glutaraldehyde-treated

tau monomer prior to adsorption to normalize for bulk

tau concentration. Filament lengths are linear under

these conditions, at least up to 10 lM bulk tau. Second,

tau can be fibrillized using anionic microspheres (which

are readily visualized in the electron microscope) instead

of fatty acids. Filaments grow from microsphere


surfaces and remain associated with them at equilib-rium. Thus interfacial microsphere concentration pro-

vides an internal standard for adsorption reactions in

the presence of competitive adsorbates such as mono-

meric tau protein. As a bonus, this strategy can be used

to normalize interfacial concentrations to the entire re-

action volume and therefore calibrate fibrillar tau with

regard to molar concentration.

Acknowledgments

This work was supported by Grant AG14452 (J.K.)from the National Institutes of Health.

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Electron microscopy as a quantitative method for investigating tau fibrillization

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