redistribution and differential extraction of soluble ... · other studies report that protein...

Journal of Cell Science 101, 731-743 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

731

Redistribution and differential extraction of soluble proteins in

permeabilized cultured cells

Implications for immunofluorescence microscopy

MELISSA A. MELAN* and GREENFIELD SLUDER

Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545, USA

*Author for correspondence

Summary

Immunofluorescence microscopy is widely used tocharacterize the cellular distribution of both soluble andstructural proteins. Control experiments generally ad-dress only the specificity of the antibodies used. Thepermeabilization/fixation conditions used to preparecells for antibody application are assumed to preservefaithfully the in vivo distibutions of the protein(s) beingexamined. We systematically tested the extent to whichsoluble proteins are redistributed into inappropriatelocations and are differentially extracted from nativelocations during the permeabilization and fixation of thecells before antibody application. We separately intro-duce six soluble FITC-conjugated proteins of differentnet charges and sizes into living cultured cells. Thelabeled proteins do not adhere to the external surfaces ofliving cells and are evenly distributed throughout thecytoplasm with the larger proteins being excluded fromthe nucleus. The cells are then prepared as if forimmunofluorescence using several conditions that en-compass many of the methods commonly used for thispurpose. Cells permeabilized with 0.1-0.2% Triton X-100 before fixation with 3.7% paraformaldehyde show a

striking localization of all but one of the test proteins tothe nucleus and/or nucleoli of 60-80% of labeled cells.Punctate cytoplasmic labeling and cytoskeletal-like ar-rays of labeled protein are also observed. Extractionwith 1% detergent prior to fixation removes most but notalways all of the exogenous proteins from the cellremnants. Permeabilization of cells with 0.1% detergentafter paraformaldehyde fixation leaves a reticular,uneven cytoplasmic distribution of the labeled proteins,and some of the larger proteins are redistributed to thenuclei. Direct fixation/permeabilization with — 20°Cmethanol largely preserves the in vivo distributions offluorescent proteins with some preferential localizationof these proteins to nuclei, nucleoli and the perinuclearregion. These results show that misleading apparentlocalizations of soluble proteins can result from theirredistribution and/or differential extraction during thepreparation of cells for primary antibody application.

Key words: fluorescence microscopy,immunocytochemistry, immunofluorescence, nucleolus,nucleus, protein localization.

Introduction

Immunofluorescence has been and continues to be animportant technique for determining the distribution ofproteins within cells. Not only has this methodologybeen used to examine the distribution of proteins thatare functionally and biochemically well characterized,but it has also been used to gain novel information onthe location, and hence function, of proteins whoseproperties are poorly understood.

In the broadest sense, preparation of cells forprimary antibody application involves membrane per-meabilization to allow entry of the antibodies into thecell and fixation to stabilize cellular structures duringsubsequent incubations and washes. In practice, threebasic approaches are used: (1) gentle detergent extrac-

tion followed by mild formaldehyde or paraformal-dehyde fixation that preserves antigenic determinants(Osborn and Weber, 1982). This procedure presumablyremoves some soluble proteins from the cells, leavingstructures that can then be visualized against a reduced'background' signal. (2) Mild formaldehyde fixationprior to detergent permeabilization (Heggeness et al.1977). This technique is intended to stabilize bothsoluble and insoluble proteins in their native locations.(3) Concurrent permeabilization and fixation by cold(-10 to —20°C) organic solvents such as methanol(Harris et al. 1980). This procedure precipitatesproteins and presumably allows the detection of bothsoluble and insoluble proteins in their native locations.

Controls for immunolocalization procedures havealmost always addressed the specificity of the primary

732 M. A. Melan and G. Sluder

antibody and the non-specific binding of the fluor-escently labeled secondary antibody to cellular com-ponents. However, an important, but generallyunstated, assumption made in all immunofluorescencestudies is that the permeabilization and fixation con-ditions used to prepare the cells for antibody appli-cation faithfully preserve the in vivo distribution of theantigen(s) of interest. This assumption may be appro-priate for polymers of proteins, such as actin andtubulin, that are known by independent criteria to formstructures that are clearly preserved by the preparationprotocols. However, this assumption may or may not bevalid for soluble proteins or proteins whose associationwith cellular structures may be transient or weak.

Assuming that the primary and secondary antibodiesare completely specific and that they faithfully displaythe distribution of the antigen of interest after fixation/extraction, there may be two important interrelatedsources of error in localizing soluble or partially solubleproteins. First, permeabilization before fixation couldallow such proteins to be redistributed into locations inwhich they are not present in vivo. Fixation of proteinsin these locations would inevitably yield misleadingresults. Second, loss of proteins from cells duringpermeabilizaton and fixation could lead to the differen-tial extraction of soluble proteins from compartmentsor structures in which they are normally present. Evenif the soluble proteins do not bind to any cellularstructures, they would appear to be preferentiallylocalized to compartments or volumes of cytoplasmfrom which they are least easily extracted. Further-more, both sources of error could operate together.These errors could also occur even when cells are lightlyfixed with monofunctional fixatives, such as formal-dehyde, before permeabilization. Unless the fixativecompletely immobilizes all soluble proteins, redistri-bution and differential extraction could lead to arti-factual distributions of the antigens.

In principle, the best way to determine the in vivodistribution of soluble proteins is by fluorescent anologcytochemistry of living cells (Wang, 1989). However,relatively large quantities of a protein must be purifiedin native form, and the derivatization of the proteinwith a chromophore must demonstrably not alter itsfunctional properties. Thus, it is not surprising thatfluorescent anolog cytochemistry of living cells has beenused in relatively few cases.

Although the potential for artifacts due to theredistribution and differential extraction of solubleproteins has not been systematically examined, therehave been indications that these errors can be apractical problem. One of the clearest examples comesfrom a comparison between the in vivo distribution offlourescently labeled gelsolin and its cellular localiz-ation as determined by immunofluorescence (Cooper etal. 1988). This study revealed a clear disparity in thelocalization of gelsolin as determined by the twomethods. Other studies report that protein distributionscan vary with the preparation technique used. Forexample, the distributions of calmodulin (Nielsen et al.1987) and of a nuclear antigen (Hoffman and Mullins,

1990) are different depending upon whether the cellsare permeabilized before or after fixation. Also,Pettijohn et al. (1984) clearly show that the NuMAantigen can exchange between cellular structures duringthe preparation of cells and isolated chromosomes forimmunofluorescence. Furthermore, the reported intra-cellular distribution of the same protein can differ fromlaboratory to laboratory. Kinesin, which is largelysoluble in vitro, has been reported to be: evenlydistributed in the cytoplasm (Hollenbeck, 1989); onmicrotubules, spindles, centrosomes, primary cilia andendoplasmic reticulum (Neighbors et al. 1988; Scholeyet al. 1985; Terasaki, 1990); or associated exclusivelywith cytoplasmic vesicles (Pfister et al. 1989).

We have sytematically investigated the extent towhich redistribution and differential extraction canproduce artifactual distributions of soluble proteinsduring the preparation of cells for immonufluorescence.Our approach has been to bead-load several types ofcultured cells with fluorescently labeled soluble proteinsof various molecular weights and net charges. We thencompared the in vivo distribution of each solubleprotein with its distibution after preparing the cells as iffor immunofluorecence microscopy using several prep-aration conditions that encompass many of the com-monly used methods.

Materials and methods

Living materialPtKj and CHO cells were maintained at 37°C in L-15(Leibovitz) medium (Sigma, St Louis, MO) buffered with 10mM Hepes and supplemented with 10% fetal bovine serum(Hazelton Biologies, Inc., Lenexa, KS). HeLa and 3T3 cellsweV* maintained at 37°C in Dulbecco's Modified Eagle'sMedium containing 5% fetal bovine serum in a 5% CO2atmosphere. All cells were grown on 22 mm X 22 mm glasscoverslips in 6-well tissue culture plates and used at sub-confluent densities.

Fluorescein-labeled protein was introduced into cells usingthe bead-loading technique of McNeil and Warder (1987).Glass beads (150-212 pan, G-9018, Sigma) were treated with 4M NaOH for 1 h, washed with distilled water until the pH ofthe water remained constant, then dried at 70°C overnight.Coverslips with cells were rinsed with PBS then flooded with35 (A of labeled protein. The cells were then covered with amonolayer of washed glass beads and agitated vigorously byhand for approximately 10 s. The glass beads were washedfrom the cells by dipping the coverslips into PBS. The cellswere allowed to recover in culture medium at 37°C for 1 h,before extraction and fixation.

Protein labelingOvalbumin (A-2512, Grade III), bovine serum albumin(BSA) (A-4503, Fraction V), /3-lactoglobulin B (L-8005),globin (G-0891), L-lactic dehydrogenase (LDH) (L-1254,Type XI), and cytochrome c (C-2506, Type HI) (all fromSigma Chemical Co., St. Louis, MO) were labeled withfluorescein-5-isothiocyanate (FITC) (F-143, MolecularProbes Inc., Eugene, OR) using a modification of theprocedures of Harlow and Lane (1988). Three mg of proteinwere dissolved in 1 ml of 0.1 M sodium carbonate, pH 9.0. Asolution of 10 mg/ml FITC in DMSO (dimethyl sulfoxide) was

Artifactual localization of soluble proteins 733

slowly added to the protein solution in samples (5 ji\ each) upto an approximately 20:1 molar ratio of dye to protein.Samples were incubated in darkness at 4°C for 18 h. Thelabeling reaction was quenched by adding NH4CI to aconcentration of 50 mM. Unbound dye was separated fromlabeled protein by gel filtration over Sephadex G-25Mcolumns (PD-10, Pharmacia, Uppsala, Sweden) using phos-phate buffered saline (PBS) for column equilibration andelution of the protein. Labeled protein in PBS was stored inamber tubes at 4°C or frozen in samples at —80°C. Proteinconcentrations were determined using a modified Lowrymicro-assay (Peterson, 1977) with BSA as the proteinstandard. Fluorescein/protein ratios were calculated fromabsorbance values at 495 ran obtained with a Beckman DU-50Spectrophotometer, and protein concentration data. Proteinsamples were diluted into 50 mM Tris-HCl, pH 8.0, beforemeasurement of the absorbance values. A molar absorptioncoefficient value for fluorescein of e=65,000 M"1 cm"' wasused in the calculations (Molecular Probes, Inc., Eugene,OR).

Gel electrophoresisFluorescein-labeled protiens were compared with their un-labeled precursors on 10% to 20% gradient SDS/polyacryl-amide gels (Laemmli, 1970). Before fixation and staining ofthe gels, fluorescein-labeled proteins were visualized on anultraviolet transilluminator (emission wavelength 302 nm,Model TM-36, UVP, Inc., San Gabriel, CA). Total proteinwas stained in the gels with Coomassie blue.

Extraction and fixation of cellsBefore fixation procedures were carried out, coverslips withattached cells were washed twice with PBS for 30 s each at25°C.

Extraction followed by fixationCells were extracted for 10 min with 0.1 M Pipes, 10 mMEGTA, 5 mM MgCl2, pH 6.9 (PEM buffer), at 37°Ccontaining Triton X-100 or Nonidet P-40 (Sigma, St. Louis,MO) at concentrations of 0.1, 0.2, 0.5 or 1%. Also, highpurity Triton X-100 (Surfact-Amps X-100, Pierce ChemicalCo., Rockford, IL) was used in some experiments at the sameconcentrations. Detergent-extracted cells were fixed for 30min at 25°C in extraction medium containing either 3.7%paraformaldehyde (Polysciences, Inc., Warrington, PA) or3.7% formaldehyde (Scientific Products, McGraw Park, IL).

Fixation before detergent permeabilizationCells were fixed with 3.7% paraformaldehyde in PEM bufferfor 30 min at 25°C and then permeabilized with 0.1% TritonX-100 in PEM for 10 min at 25°C.

Solvent fixation/permeabilizationCells were directly fixed and permeabilized in 90% methanol,50 mM EGTA, pH 6.0, for 5 min at -20°C (Osborn andWeber, 1982).

Fixation followed by solvent extractionCells were fixed with 3.7% paraformaldehyde in PEM for 30min at 25°C and then permeabilized in either absolute acetoneat -20°C or 90% methanol, 50 mM EGTA, pH 6.0, at -20°Cfor 5 min.

After the fixation/permeabilization regime, the coverslipswere rinsed with PBS and mounted onto slides with apolyvinyl alcohol mounting medium (Osborn and Weber,1982) containing 2.5% l,4-diazobicyclo-(2,2,2)-octane to

reduce photobleaching of the fluorescein (Johnson et al.1982).

BSA immunofluorescenceFollowing fixation and permeabilization (as above), the cellswere blocked with 1% ovalbumin (Calbiochem, San Diego,CA) in PBS for 10 min. Cells were then exposed to a 1:500dilution of mouse monoclonal anti-bovine serum albumin(Sigma, St. Louis, MO) for 10 min followed by treatment witha 1:100 dilution of fluorescein-labeled sheep anti-mouse IgG(Sigma, St. Louis, MO) for 10 min. Antibodies were dilutedwith PBS containing 1% ovalbumin. Coverslips weremounted as described above.

Fluorescence microscopySlides were observed with a Zeiss Axioskop (Carl Zeiss, Inc.,Thornwood, NY) equipped for both phase-contrast and epi-fluorescence microscopy. A narrow bandpass filter set (CarlZeiss, Inc., Thornwood, NY) was used for fluoresceinobservations. Photographs were taken with a Zeiss MC100camera (automatic exposure) on Kodak T-Max 400 film anddeveloped in Kodak D-76 developer (Eastman Kodak,Rochester, NY).

To obtain the quantitative data shown in Table 2 (below),we scored only cells that showed fluorescent labeling. Sinceany given cell could be scored in more than one category, thesum of the percentages for any one protein may be greaterthan 100%.

Results

We tested the cellular distribution of soluble proteinsafter preparing cells as if for immunofluorescence byseveral commonly used approaches: detergent extrac-tion followed by formaldehyde fixation, formaldehydefixation followed by detergent permeabilization, anddirect permeabilization/fixation with cold methanol.Given the extremely numerous slight modifications ofthese basic themes used by various workers, we did notattempt to duplicate precisely the conditions used in allprevious studies. Instead, we used conditions thatencompassed the particular methods that have beencommonly used.

Protein labeling and characterizationTable 1 lists the properties of the protein reagents thatwere individually bead-loaded into cells. Protein sol-utions were applied extracellularly at the concen-trations indicated. The electrophoretic patterns of thethe FITC-labeled proteins are shown in Fig. 1A. Thefluorescence of the labeled ovalbumin (lane 1, asterisk)is barely visible on the gel due to the low level of FITCbinding to this protein (F/P=0.47). The minor bandsvisible in some lanes are probably due to contaminantsin the starting preparations of proteins. Fig. IB shows aCoomassie-stained gel of labeled and unlabeled pro-teins run in adjacent lanes. All of the labeled proteins(odd-numbered lanes) show a slight shift to a higherrelative mobility. The minor high molecular weightband seen in the globin samples (lanes 7 and 8) may bedue to serum albumin impurity in the sample.


Table 1. Fluorescein-labeled protein samples

Protein

OvalbuminBSA/3-Lactoglobulin BGlobin

Lactate dehydrogenase

Cytochrome c

yWr(xl(T3)(unmodified protein)

456618.462

la chains (15.0)2/3 chains (15.9)

1404 subunits (36)

12.2

Isoelectricpoint

(unmodifiedprotein)

4.64.95.16.8

8.5

9.6

Concentration(mg/ml)

0.970.910.621.05

0.43

0.91

Fluorescein/protein

ratio

0.472.231.142.55

6.12

1.49

1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12

66-

Fig. 1. SDS-polyacrylamide gel electrophoresis of FTTC-labeled and native proteins. (A) FTTC-conjugated proteinsviewed with an ultraviolet transilluminator (A=302 nm).Lanes 1-6, ovalbumin, BSA, /S-lactoglobulin B, globin,LDH and cytochrome c, respectively. Asterisk marks theband of ovalbumin, which is barely visible. (B) Coomassieblue-stained gel of FTTC-labeled protein and native proteinrun in adjacent lanes. Lanes 1-2, ovalbumin; 3-4, BSA; 5-6, /3-lactoglobulin B; 7-8, globin; 9-10, LDH; 11-12,cytochrome c. Relative molecular mass markers (xlO~3)are indicated to the left of A. Five fig protein/lane.

Distributions of labeled protein in living cellsDuring the bead-loading procedure, the living cells areexposed to labeled proteins for 0.5-1 min. To test forpossible binding of the labeled proteins to external cellsurfaces, living PtKi cells were exposed to the fluor-escent proteins for 10-30 min and then rinsed with PBSprior to observation in vivo on the fluorescencemicroscope. Fig. 2A-A' shows phase-contrast andfluorescence micrographs of a group of cells after a 10min exposure to labeled cytochrome c. Fig. 2B-B'shows another field of cells following exposure to FITC-globin for 20 min. For all tests, either no fluorescencewas observed or a very low level of fluorescence wasseen. The low level of fluorescence shown here has beenaccentuated by the long photographic exposures due tothe automatic exposure control of the camera. Theseobservations indicate that live PtKi cells do not bindsignificant amounts of FITC-labeled proteins to their

external surfaces during the 0.5-1 min duration of thebead-loading procedure. After greater than 30 minexposure to labeled proteins in the medium, some cellscontained punctate fluorescence in the perinuclearregion, suggesting pinocytotic uptake (data not shown).

Following bead-loading, individual cells showedvarying levels of protein incorporation (Fig. 2C-C).The variable quantity of protein incorporated afterbead-loading occurred for all proteins in all the celltypes that we examined and is consistent with thefindings of McNeil and Warder (1987).

Fig. 3 A-F shows representitive examples of the invivo distributions of the 6 FITC-labeled proteins inliving cells. Proteins with MT values greater than~50,000 were evenly distributed throughout the cyto-plasm but excluded from the nucleus. Proteins with MTvalues less than ~50,000 were distributed throughoutboth the cytoplasm and the nucleus. The in vivodistribution of globin sometimes showed two types oflocalization (Table 2; Fig. 3D inset). The nuclearlocalization of FITC-globin may reflect the dissociationof some globin molecules into subunits that are smallerthan 50,000 MT. In no case did we observe preferentiallocalization of labeled proteins into linear cytoplasmicarrays, perinuclear spots or nucleoli (Table 2).

Distributions of labeled protein in cells extracted withdetergent before fixationWe extracted interphase PtKi cells loaded with FITC-labeled proteins with Triton X-100 or Nonidet P-40before fixation with either formaldehyde or paraformal-dehyde. We used a range of detergent concentrationsthat include those found in many published studies:0.1% (Bonifacino et al. 1985; Goldenthal et al. 1985;Hoffman and Mullins, 1990), 0.2% (Osborn andWeber, 1982; Vandr6 et al. 1984; Vandr6 et al. 1986)and 1% (Balczon and Schatten, 1983; Hollenbeck,1989). As a standard, we used a 10 min extraction time.Published extraction times range from 1 to 60 min, andour control experiments using longer (20-30 min) orshorter (2-5 min) extraction times revealed that theresults shown below are not qualitatively sensitive tovariations in extraction duration (data not shown).

Extraction with 0.1% detergent prior to fixationFluorescent protein distributions in interphase PtKx


B

Fig. 2. (A,A') Phase-contrast and fluorescence micrographs of living PtK] cells treated with 0.91 mg/ml FTTC-cytochrome cfor 10 min without bead-loading. (B,B') Phase-contrast and fluorescence micrographs of living PtK! cells treated with 1.05mg/ml FITC-globin for 20 min without bead-loading. Note low fluorescence in A' and B', indicating a lack of binding tothe cell surfaces. (C,C) Phase-contrast and fluorescence micrographs of a field of living PtK! cells bead-loaded with FITC-globin. Cells were exposed to external labeled protein for <1 min. Variations in the levels of fluorescence intensity indicatedifferent amounts of protein loaded into the cells. 10 ftm per scale division (black bars in all Figs).

Fig. 3. FTTC-protein distributions in living PtKl cells. (A- F) Ovalbumin, BSA, /3-lactoglobulin B, globin, LDH andcytochrome c, respectively. Proteins are distributed evenly throughout the cells with proteins of Mr greater than 50,000excluded from the nuclei (B,D,E). In some cases, globin also exhibits nuclear fluorescence (D, inset). 10 jjm per scaledivision.Fig. 4. FITC-protein distributions in PtKi cells following a 10 min extraction with 0.1% Triton-X 100 and 30 min fixationwith 3.7% paraformaldehyde. (A-F) Ovalbumin, BSA, /3-lactoglobulin B, globin, LDH and cytochrome c, respectively. Allproteins except cytochrome c (F) show retention within the cells. The greatest fluorescence intensities are seen in the nucleiand nucleoli. A low percentage of cells (2%) loaded with FTTC-BSA (B) show a pattern of fluorescence in the cytoplasmindicative of cytoskeletal association. Arrows in C, D, and E indicate juxtanuclear localizations. 10 fan per scale division.

cells that are extracted for 10 min with 0.1% Triton X-100 or Nonidet P-40 prior to fixation are shown in Fig. 4A-F. For cells loaded with cytochrome c, the smallest ofthe test proteins, such extraction removes essentially allthe labeled protein from the nucleus and cytoplasm(Fig. 4F and Table 2). For cells loaded with the other

test proteins, extraction removes much, but not all, ofthe fluorescent probes from the cytoplasm leavingdiffuse, reticulate or linear cytoplasmic arrays offluorescent material in approximately half of the loadedcells (Table 2). In 2% of the cells labeled with BSA, theprotein appears to be associated with linear cytoskeletal


Table 2. Frequency of localization patterns (%

Procedure

In vivo distributions

Protein

OvalbuminBSA/J-Lactoglobulin BGlobinLDHCytochrome c

Detergent extraction before fixation0.1% Detergent

0.2% Detergent

1% Detergent

Fixation before extraction3.7% Paraformaldehyde/0.1% Detergent

Solvent fixation/permeabilization-20°C Methano!

•Linear cytoplasmic elements;

OvalbuminBSA/3-Lactoglobulin BGlobinLDHCytochrome cOvalbuminBSA^-Lactoglobulin BGlobinLDHCytochrome cOvalbuminBSA/3-Lactoglobulin BGlobinLDHCytochrome c

OvalbuminBSA/S-Lactoglobulin BGlobinLDHCytochrome c

OvalbuminBSA^-Lactoglobulin BGlobinLDHCytochrome c

tjuxtanuclear spots;

Cytoplasmic

100100100100100100

5248634759

34501924

2512

889793919494

959795999694

tprimary cilia.

%(single cells showing 1

are scored in

Localizationocalization in multiple structuresmore than one category)

Nuclear Nucleolar

1000

100580

100

6880756974

No fluorescenceNo fluorescence

60732662

No fluorescenceNo fluorescenceNo fluorescence

2823

No fluorescenceNo fluorescence

453548544955

62

1230

317

1000

100580

100

108

554946

detecteddetected

5346522

detecteddetecteddetected

9496

detecteddetected

171394

50

01

1638

122

Other

000000

02*It0.5t

16t

2*2t4t, 2t2t

00

4t6t7t

30t8t9t

0It0000

Cells scored (n)

524461794355

127188172203142

131219118101

110103

12898

120105114105

9214312911711798

arrays (Fig. 4B). Also, small percentages of cells showlocalization of /3-lactoglobulin B, globin and LDH to asingle focus next to the nucleus (Table 2 and Fig. 4C-E,arrows).

With the exception of cytochrome c, all labeledproteins are strikingly localized in the nucleus and/ornucleoli of 68-80% of the loaded cells (Fig. 4A-E andTable 2). BSA, however, generally was not redisributedto the nucleoli (Fig. 4B and Table 2), and preferentiallocalization of other proteins, such as globin (Fig. 4D),to nucleoli was observed at a lower frequency than fornuclear localization (Table 2).

We established the percentage of label retention invarious cellular domains only in cells that showed atleast some fluorescence (Table 2). Thus, these datashow the relative frequency of the different categories

of label distribution. Cells loaded with small amounts oflabeled protein and cells from which the label had beencompletely extracted are not scored. In this regard,however, we did not observe any obvious, systematicreduction in the total percentage of cells in a prep-aration that were labeled after detergent extraction,except where indicated in Table 2.

The fluorescence in the detergent-extracted cellsshown here and in other experiments is not caused byfluorescent impurities in either the fixative or detergent.Control extractions/fixations conducted on cells thatwere not loaded with FITC-labeled protein showedneither background fluorescence nor localized fluor-escence. Furthermore, we found no difference instructural preservation and/or fluorescence distributionwhen reagent grade Triton X-100 or formaldehyde was

used and no difference in fluorescence distribution ifNonidet P-40 was used in place of Triton X-100.

Extraction with 0.2% detergent prior to fixationFluorescent protein distributions in interphase PtKicells that are extracted for 10 min with 0.2% Triton X-100 or NP-40 prior to fixation are shown in Fig. 5 A-F.For cells loaded with cytochrome c and ovalbumin, thisextraction removes essentially all the the labeledprotein from the nucleus and cytoplasm (Fig. 5A, 5Fand Table 2). For cells loaded with the other testproteins, labeled proteins were localized to nuclei andnucleoli (Fig. 5 B-E). There is a low but finite incidenceof labeled protein localization to linear arrays in thecytoplasm reminiscent of microtubules (seen only withBSA) and to single juxtanuclear spots (Fig. 5B). In 4%of the cells loaded with FITC-globin, we observed labellocalization to a single linear element on each cell,which may be the primary cilium (Fig. 5D, lower inset).The higher percentage of cells that show nucleolarlocalization of globin relative to corresponding cellsextracted with 0.1% detergent may reflect the higher


contrast of nucleolar label against a reduced back-ground of nuclear label.

Extraction with 1 % detergent prior to fixationFluorescent protein distributions in interphase PtKicells that are extracted for 10 min with 1% Triton X-100prior to fixation are shown in Fig. 6 A-F. In general, thehigher detergent concentration leads to a noticeablygreater extraction of all test proteins. Essentially allovalbumin, BSA, LDH and cytochrome c are removedfrom both cytoplasmic and nuclear domains (Fig.6A,B,E,F and Table 2). Only cells loaded with £-lactoglobulin B and globin showed retention of somelabel in cytoplasmic, nuclear and nucleolar domains(Fig. 6C-D). These labeled proteins were most fre-quently retained in the nucleoli (Table 2).

Fixation before permeabilizationWe fixed living PtKj cells for 30 min with 3.7%paraformaldehyde, then extracted them with 0.1%Triton X-100 according to the methods outlined byCooper et al. (1988) and Hoffmann and Mullins (1990).

Fig. 5. FTTC-protein distributions in PtK! cells following a 10 min extraction with 0.2% Triton-X 100 and a 30 min fixationwith 3.7% paraformaldehyde. (A-F) Ovalbumin, BSA, ^-lactoglobulin B, globin, LDH and cytochrome c, respectively.Ovalbumin (A) and cytochrome c (F) are completely removed from the cells. The remainder of proteins show stronglocalization in the nuclei and/or nucleoli. Approximately 2% of cells loaded with FTTC-BSA (B) show possible cytoskeletalassociation. Cells loaded with FTTC-globin (D) show a variety of localizations including nucleoli and centrosomes (upperinset) and primary cilia (lower inset). Arrows in B and D (upper inset) indicate juxtanuclear localization. 10 jim per scaledivision.Fig. 6. FITC-protein distributions in PtKt cells following a 10 min extraction with 1% Triton X-100 and 30 min fixationwith 3.7% paraformaldehyde. (A-F) Ovalbumin, BSA, /?-lactoglobulin B, globin, LDH and cytochrome c, respectively. Allproteins except /3-lactoglobulin B (C) and globin (D) are removed from the cells. The strongest fluorescent signals are seenexclusively in the nucleoli with some cells showing retention of label in the nucleus as well (D, inset). 10 /mi per scaledivision.


Fig. 7. FITC-protein distributions in PtKL cells fixed for 30 min with 3.7% paraformaldehyde then permeabilized with 0.1%Triton X-100 for 10 min. (A-F) Ovalbumin, BSA, /3-lactoglobulin B, globin, LDH and cytochrome c, respectively. Allproteins show a reticulate cytoplasmic distribution. Cells loaded with FITC-BSA, globin and LDH display nuclearfluorescence in contrast to the in vivo exclusion of these proteins from the nuclei. The cell-to-cell variability of thistechnique is shown in D. Possible association of label with the interphase centrosome was frequently seen with thistechnique (arrows, A, C, D and F). 10 //m per scale division.

For FITC-conjugated ovalbumin, /Mactoglobulin B andcytochrome c (Fig. 7A, C and F, respectively) fluor-escence distribution after fixation and extraction re-sembled the in vivo distribution, with two exceptions:the cytoplasmic domains had a coarser, more reticulateappearance and 4-9% of the labeled cells showed asingle bright spot adjacent to the nucleus (7A, C and F,arrows and Table 2). The position and number of theseloci were confirmed by focusing through the cell.

The cytoplasmic distribution of FITC-conjugatedBSA, globin and LDH also appeared to be morereticulate than in vivo (Fig. 7B, D and E, respectively)and was sometimes noticeably punctate. Six to 30% ofthe labeled cells showed a single spot next to thenucleus in the expected location of the centrosome. In35 and 49% of the cells loaded with BSA and LDH,respectively, we observe a striking relocalization oflabeled proteins to the nucleus (Table 2). Labeled BSArelocalized to and was retained in the nuclear matrix butnot in the nucleoli (Fig. 7B).

Permeabilization of membranes following chemicalfixation can also be accomplished by using organic

solvents in place of detergents (Osborn and Weber,1982; Baron and Salisbury, 1988). We conducted sevenseparate trials in which we permeabilized cells witheither methanol or acetone after fixation. In allexperiments, the remaining fluorescent signal was lowand consequently we were unable to determine thedistribution of remaining labeled proteins, if any,within the cell remnants (data not shown).

Direct fixation/permeabilization with cold methanolCells were loaded with FITC-protein and then directlyfixed/permeabilized with 90% methanol containing 50mM EGTA at -20°C (Osborn and Weber, 1982). Ingeneral, the distributions of labeled proteins approxi-mated, but did not duplicate, the corresponding proteindistributions in vivo (Fig. 3A-F vs Fig. 8A-F). Thecytoplasmic distribution of fluorescence exhibited afibrous, reticulate texture not observed in vivo and theoverall fluorescent intensity of the population of fixedcells was significantly lower than in vivo. Largerproteins for the most part remained excluded from thenucleus; however, cytoplasmic fluorescence above and

Fig. 8. FITC-protein distributions in PtK! cells after fixation for 5 min in 90% methanol, 50 mM EGTA at -20°C. (A-F)Ovalbumin, BSA, /S-lactoglobulin B, globin, LDH and cytochrome c, respectively. All proteins show a fibrous texturedfluorescence in the cytoplasm and stronger fluorescence at the periphery of the nucleus. Globin (D) and cytochrome c (F)also show fluorescence in the nucleoli. The overall level of fluorescence for all proteins is lower than that seen in vivo. 10/an per scale division.


below the nucleus sometimes made this determinationdifficult (Fig. 8B and E). In many cells, the fluorescencewas greater in the region surrounding the nucleus thanin the peripheral cytoplasm (Fig. 8C,D and E). Thismay be due to the greater cytoplasmic thickness in theseregions coupled with the partial extraction of the testproteins from the cytoplasm. In addition, the proteins/J-lactoglobulin B, globin and cytochrome c appear tobe preferentially retained in the nucleoli (Table 2).

Localization of underivitized BSAWe tested the formal possibility that the distributions oflabeled proteins shown in Figs 4-8 could be due partiallyor totally to peculiar properties of these proteins causedby derivitization with FTTC. Thus, we bead-loadedunmodified BSA (0.91 mg/ml in PBS) into PtKx cellsand processed them in the same ways as the cells loadedwith FITC-proteins. We then used indirect immunoflu-orescence to determine the distribution of the unmodi-fied BSA. Fig. 9A, B and C, respectively, show cellsthat were extracted with 0 .1%, 0.2% and 1% detergentprior to formaldehyde fixation. The distributions ofunmodified BSA match those of the FITC-BSA (seeFigs 4B, 5B and 6B). Fig. 9D shows the distribution ofunlabeled BSA in a cell that was fixed with paraformal-dehyde before extraction with 0.1% detergent (com-pare with the FITC-BSA distribution shown in Fig. 7B).The distribution of unlabeled BSA in a cell that wasfixed/permeabilized with cold methanol is shown in Fig.9E (compare with the FITC-BSA distribution shown inFig. 8B). The higher cytoplasmic fluorescence of cellsprepared by indirect immunofluorescence compared tothose loaded with FITC-BSA (Fig. 9E vs Fig. 8B) mayrepresent signal amplification due to the binding ofmultiple primary and secondary antibodies.

Protein localizations in different cell typesTo determine if the results shown above are unique toPtKi cells, parallel experiments were conducted withCHO, 3T3 and HeLa cells bead-loaded with FITC-BSAas the test protein. In living cells the FITC-BSA isuniformly distributed throughout the cytoplasm and isexcluded from the nucleus in all cell types (Fig. 10 A-D).

The distribution of FITC-BSA in cells extracted with0.1% detergent and then fixed with formaldehyde isshown in Fig. 10 E-H. In PtKa cells, labeled BSA is seenprimarily in the nucleus (excluding the nucleoli) withsome retention in the cytoplasm. For CHO cells, thisextraction removes almost all of the tagged protein;only a very weak cytoplasmic signal can be detected insome cells (Fig. 10 F). In 3T3 cells the FITC-BSA isseen in the nucleus and possibly the nucleoli; labeledprotein is generally completely extracted from thecytoplasm (Fig. 10 G). HeLa cells generally show weakcytoplasmic fluorescence with some of the labeledprotein in the nucleus (Fig. 10 H).

The distribution of FITC-BSA in cells extracted with1% detergent and then fixed with formaldehyde, isshown in Fig. 10 I-L. With the exception of label in thenuclei of a low percentage of the HeLa cells, this regimeextracts the labeled probe from all cell types.

The localization of FITC-BSA in cells fixed withparaformaldehyde before permeabilization with 0.1%Triton X-100 is shown in Fig. 10 M-P. For all cell types,the cytoplasmic distribution of label has a coarse,fibrous appearance with areas devoid of label. This is inclear contrast to the label distribution in living cellswhere the loaded protein is uniformly dispersedthroughout the cytoplasm. In CHO cells there is astriking relocalization of FITC-BSA to the nucleus butnot the nucleoli (Fig. 10 N). This same relocalization

Fig. 9. Distribution of underivatized BSA in PtKi cells subjected to various preparation procedures. (A-C) Cells extractedfor 10 min with Triton X-100 before fixation for 30 min with 3.7% formaldehyde. (A) Extraction with 0.1% detergent.BSA is detected in the nucleus and cytoplasm but not in the nucleoli. (B) Extraction with 0.2% detergent. Faintfluorescence is seen in the cytoplasm. (C) Extraction with 1% detergent. No fluorescence detected. (D) Cell fixed for 30min with 3.7% paraformaldehyde before permeabilization for 10 min with 0.1% Triton X-100. Fluorescence is seenprimarily in the cytoplasm with a low level of fluorescence seen in the nucleus but not the nucleoli. (E) Cell fixed for 5 minin 90% methanol, 50 mM EGTA at —20°C. Fluorescence in the cytoplasm shows a fibrous texture. BSA localized in cellswith monoclonal mouse anti-BSA antibodies and FITC-conjugated secondary antibody. 10 jan per scale division.

740 M. A. Melon and G. Sluder

occurs in PtKj cells as well, but to a lesser extent (Fig.10 M).

The distribution of FITC-BSA after direct fixation/permeabilization with methanol at -20°C is shown inFig. 10 Q-T. For all cell types the cytoplasmicdistribution of the labeled protein has a coarse fibroustexture and an overall low fluorescence intensity. Thelabel is not redistributed to the nuclei of PtKx and HeLacells but is to a limited extent in CHO and 3T3 cells

Fig. 10. Distribution of FITC-BSA in various cell lines.(A- D) Protein distribution inliving cells, PtK1; CHO, 3T3and HeLa cells, respectively.The protein is excluded fromthe nuclei of all cells.(E-H) Protein distribution incells extracted for 10 min with0.1% Triton X-100 beforefixation for 30 min with 3.7%formaldehyde, PtK^ CHO,3T3 and HeLa cells,respectively. Nuclearfluorescence is seen in PtK:(E) and 3T3 (G) cells.(I-L) Protein distribution incells extracted for 10 min with1% Triton X-100 beforefixation for 30 min with 3.7%formaldehyde, PtK1; CHO,3T3 and HeLa cells,respectively. No fluorescence isdetected in the cells with theexception of some nuclearfluorescence seen in HeLa cells(L). (M-P) Protein distributionin cells fixed for 30 min with3.7% paraformaldehyde beforepermeabilization for 10 minwith 0.1% Triton X-100.Fluorescence is seen primarilyin the cytoplasm with theexception that nuclearfluorescence is seen in PtKL(M) and CHO (N) cells.(Q-T) Protein distributions incells fixed for 5 min with 90%methanol, 50 mM EGTA at-20°C, PtKj, CHO, 3T3 andHeLa cells, respectively. Allcells show an overall lowfluorescence, fibrous texturedcytoplasmic fluorescence andbright staining at the peripheryof the nucleus. 10 fim per scaledivision (black bar).

(Fig. 10 R and S). In addition, this technique enucleatesmost of the 3T3 cells (data not shown).

Global redistribution of labeled proteinWe often observed that low levels of fluorescence werepresent in the cytoplasm of all cells on the coverslip forall of the techniques and for all cell lines used in thisstudy. This is not due to uniform loading of cells, as


demonstrated by our in vivo observations (Fig. 2C), orto autofluorescence of the detergent or fixative. Thisglobal fluorescence suggests that label extracted fromloaded cells diffuses to and becomes trapped in othercell remnants during extraction and fixation.

Discussion

Immunofluorescence microscopy has been used todetermine the cellular localization of proteins in orderto gain insight into their function. This approach hasbeen particularly important for studies on proteins thatare poorly understood. However, any conclusion aboutthe localization of a protein, and hence its presumedfunction, critically depends upon the faithful preser-vation of its in vivo distribution. Any redistribution ordifferential extraction of the antigen of interest duringthe preparation of the cells for immunofluorescencecould suggest interactions or functions that are incor-rect.

To provide the clearest possible test for the redistri-bution and differential extraction of proteins, weseparately introduced six FITC-labeled proteins intocultured cells and directly compared their in vivolocalizations with their distributions after permeabiliz-ation and fixation as if for immunofluorescence. Directlabeling of the proteins allowed us to avoid questionsabout antibody specificity and non-specific binding ofsecondary antibodies to cellular components. In ad-dition, four of the six test proteins should not normallybe present in these cells; this reduces the possibility ofspecific or physiologically relevant binding of the testproteins to cellular components.

Detergent extraction before fixationThis protocol is often used to remove soluble proteinsand the unpolymerized pools of cytoskeletal proteinsprior to fixation and application of the primaryantibody. Given that we loaded the cells with solubleproteins that should not have physiologically relevantinteractions with cellular components, we could haveexpected the test proteins to have been completelyremoved from the cell remnants, or reduced butremaining in the in vivo locations. Our results show, onthe contrary, that extraction of cells with nonionicdetergents before fixation with monofunctional alde-hydes can lead to both the redistribution and differen-tial extraction of soluble proteins producing apparentlocalizations that are entirely artifactual. The redistri-bution of soluble proteins is most clearly demonstratedin cells loaded with the higher Mr proteins, such asBSA, and extracted with 0.1% or 0.2% detergent. Inliving cells, these proteins are uniformly distributed inthe cytoplasm but excluded from the nucleus. Afterextraction and fixation, they are strikingly localized inthe nucleus and/or nucleoli in the majority of thelabeled cells. In addition, extraction of the labeledproteins from the cytoplasm was not complete oruniform; we observed label in cytoskeletal-like arraysand in a single spot next to the nucleus, which could bethe remnant of the interphase centrosome.

These results are not due to the fluorescein moiety,which conceivably might impart unusual properties tothe test probes that would cause them to be distributedin a qualitatively different fashion than native proteins.Indirect immunofluorescence of cells loaded with nativeBSA showed in all cases that the distribution of nativeBSA was the same as that of derivatized BSA. Inaddition, control experiments show that the distributionof fluorescence could not be due to the fixatives ordetergents themselves or to impurities in these re-agents.

We found that the extent of the extraction of labeledproteins was a function of detergent concentration,protein size, protein charge, cellular structure and celltype. Cytochrome c, a relatively small and basicprotein, is essentially completely extracted from boththe cytoplasmic and nuclear compartments by alldetergent extractions used. For larger proteins, increas-ing detergent concentration reduced but did not alwayseliminate the residual labeled protein found in both thenucleus and cytoplasm.

Our comparison of the distribution of labeled BSA indifferent cell types shows that the extent of extraction ofthis protein can also vary between cell types. Forexample, at low detergent concentrations (0.1%) thelabel is noticably more extracted from CHO and HeLacells than from PtKx and 3T3 cells. Extraction with 1%detergent, however, removes most of the labeled BSAfrom all cell types.

Fixation before permeabilizationIn principle, fixation with paraformaldehyde prior topermeabilization should faithfully preserve the in vivodistribution of all proteins by fixing them in place beforethe cell is permeabilized with detergent. However, ourresults reveal that redistribution and differential extrac-tion of soluble proteins can occur in cells prepared bythis technique. We found that the cytoplasmic distri-bution of the labeled proteins had a coarse, punctateand sometimes reticulate appearance that differed fromthe uniform in vivo distribution. Depending on theparticular protein, between 4 and 30% of the labeledcells exhibited a single fluorescent spot next to thenucleus, which may represent preferential retention ofprobe in the remnant of the centrosome. Importantly,for greater than 35% of the cells loaded with labeledBSA, globin and LDH, we observe a relocalization oflabeled proteins to the nucleus.

We also found that the extent of the discrepancybetween the in vivo and post-preparation distributionsof a soluble protein can vary between cell types. Thedistribution of labeled BSA in fixed/permeabihzed 3T3and HeLa cells showed the closest resemblance to the invivo distribution. However, in PtKx and CHO cells, thecytoplasmic distribution of labeled BSA was noticablycoarser and there was clear evidence for redistributionof the probe into the nucleus where it is not found invivo.

Direct fixation/permeabilization with cold methanolThis protocol is commonly assumed to precipitate


completely both structural and soluble proteins in placewithout loss, thereby faithfully representing the in vivodistribution. Our results show that the distributions oflabeled proteins did not duplicate the correspondingprotein distributions in vivo. The cytoplasmic distri-bution of fluorescence exhibited a fibrous, reticulatetexture not observed in living cells and the overallfluorescence intensity of the populations of fixed cellswas lower than the in vivo fluorescence. This reductionin fluorescence intensity may be due to the partialextraction of proteins (Fujiwara and Pollard, 1980). Apractical consequence of this partial extraction, forsome cell types, is that the antigen of interest canappear to be preferentially localized around the nucleusand not uniformly distributed throughout the cytoplasmas it was in vivo. In addition, for some proteins such aslabeled cytochrome c and globin, this protocol leads tothe apparent preferential retention of the proteins inthe nucleoli. There is also a low but finite redistributionof higher Mr proteins into the nuclear and nucleolarcompartments.

ConclusionsOur results demonstrate that none of the permeabiliz-ation/fixation methods we tested completely preservesthe in vivo distribution of soluble proteins. Thesemethods commonly used to prepare cells for immuno-fluorescence can lead to the redistribution and differen-tial extraction of soluble proteins before antibodyapplication, thereby producing misleading patterns forthe localization of such proteins. If indirect immunoflu-orescence is subsequently used, possible signal amplifi-cation for artifactually distributed proteins could maketheir localizations appear striking and, therefore,subjectively real. Since these artifacts will be super-imposed upon the real and physiologically relevantdistributions of the proteins of interest, the interpe-tation of immunofluorescent images is bound to bedifficult. Unless the problems of redistribution anddifferential extraction can be demonstrably excluded,common sense must be used in matching the particularprotocol used to prepare cells for immunofluorescencewith the expected properties of the protein beingexamined and the sort of information being sought.Also, images should be evaluated with the realizationthat partial and differential extraction of the antigen aredefinite possibilities.

While use of the bifunctional cross-linking fixativeglutaraldehyde should produce superior antigen immo-bilization, this fixative is not commonly used forimmunofluorescence, due to non-specific backgroundfluorescence and/or loss of antigenicity (Nakane, 1975;Cande et al. 1977; Weber et al. 1978). Our attempts todemonstrate the fixation of soluble proteins withglutaraldehyde resulted in very high levels of overallfluorescence even after treatment of the cells withsodium borohydride (Weber et al. 1978). Nonetheless,DeBrabender et al. (1977) used glutaraldehyde fixationto show effectively the localization of the soluble poolof tubulin in cells treated with colchicine or vinblastine.However, they localized the protein within the cells

with peroxidase-linked antibodies and a precipitationreaction visualized with bright-field microscopy in placeof fluorescence observations. In addition, Rieder andAlexander (1990) elegantly localized individual micro-tubules in glutaraldehyde-fixed cells with an antibodythat fortuitously recognized glutaraldehyde-treatedtubulin. This raises the intriguing possibility thatprimary antibodies raised against glutaraldehyde-treated antigens in conjunction with peroxidase-labeledsecondary antibodies may prove to be a better means oflocalizing soluble proteins than the immunofluor-escence techniques that are currently in use.

Finally, our observations emphasize the obviousimportance of using other approaches to determineindependently the properties and functional interac-tions of proteins with recognized cellular structures.

We thank Drs. Samuel S. Bowser, Yu Li Wang and Earl F.Baril for providing the cell lines used in this investigation. Wealso thank Dr. Martin L. Jones for generously donating cellculture equipment and Mr. Fredrick J. Miller for photo-graphic assistance. We especially thank Drs. Yu Li Wang, D.Lansing Taylor and Richard A. Cardullo for critical evalu-ation of the manuscript and valuable suggestions. This workwas supported by NIH GM-30758 to G. Sluder and NCI PO-30-12708 to the Worcester Foundation for ExperimentalBiology.

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(Received 28 October 1991 - Accepted, in revised form,15 January 1992)

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