Subcellular Localization of Alkaline Phosphatase in Bacillus

JOURNAL OF BACTERIOLOGY, Aug. 1984, p. 668-6770021-9193/84/080668-10$02.00/0Copyright © 1984, American Society for Microbiology

Vol. 159, No. 2

Subcellular Localization of Alkaline Phosphatase in Bacilluslicheniformis 749/C by Immunoelectron Microscopy with Colloidal

GoldGUAN TINGLU, ARATI GHOSH, AND BIJAN K. GHOSH*

Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Rutgers Medical School,Piscataway, New Jersev 08854

Received 18 November 1983/Accepted 1 May 1984

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by animmunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidalgold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane andcytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in associationwith the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a fewclusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled,frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for thealkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membranesuggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B.licheniformis 749/C.

Alkaline phosphatase is a secretory protein in Bacilluislicheniformis (6, 8; B. K. Ghosh and A. Ghosh, in B. K.Ghosh, ed., Organization ofProkarvotic Cell Membrane, inpress). In actively growing cells, 30 to 50% of the enzyme issecreted. Cell fractionation and ultrastructural cytochemis-try have shown that the major amount of cell-bound alkalinephosphatase is distributed in the cell'envelope (i.e., theplasma membrane and cell wall) (8). However, it is notknown whether there is any precursor-product relationshipbetween the bound and secreted alkaline phosphatase of theplasma membrane. Recently, it has been demonstrated thatthe alkaline phosphatase of B. licheniformis is synthesized ina precursor form (Nelson et al., submitted for publication);hence, nascent alkaline phosphatase molecules are likely tocontain a signal sequence. It has been shown in animal cellsthat the signal sequence interacts with a specific region in themembrane which is defined as the secretory apparatus (25).Although direct proof for the existence of such a secretoryapparatus is not available in bacteria, indirect genetic evi-dence suggests the possible existence of a plasma mem-brane-bound secretory apparatus (17; Ghosh and Ghosh, inpress). It has been suggested that Bayer junctions (1) aresecretory sites in gram-negative cells. We assumed that theputative secretory apparatus in the plasma membrane of B.licheniformis would bind alkaline phosphatase which wouldcause its eventual secretion.We have determined the subcellular distribution of alka-

line phosphatase with a probe consisting of anti-alkalinephosphatase antibody labeled with colloidal gold particles(i.e., gold-immunoglobulin G [IgG] complex), which havebeen widely used as markers for specific 'detection of mem-brane receptors (4, 12, 14). Colloidal gold has advantagesover ferritin, such as (i) high electron density, (ii) minimalnonspecific aggregation, and (iii) low nonspecific binding. Toprevent denaturation of the antigenicity of alkaline phospha-tase, frozen sections were primarily used for labeling. Thelabeled sections were analyzed both qualitatively and quanti-tatively. Differential distribution in subcellular structures or

* Corresponding author.

subtle changes in the subcellular distribution in relation tophysiological conditions cannot be assessed unless bindingof the gold-IgG particles to different samples can be quantita-tively evaluated. The problem of generating quantitativedata from electron micrographs is a long-standing one.Quantitative measurements made on electron micrographscan be statistically analyzed. However, standard statisticalevaluation (based on numerical probability) requires mea-surements from an enormously large number of samples toobtain a significant sample size, because the number ofbacterial cells on micrographs represents a minor fraction ofthe vast population of bacteria in growth media. Weibel hasapplied stereology to avoid this problem (26, 27). Thisquantitative technique is used extensively for the analysis ofelectron micrographs of animal cells. We have introducedthe application of this technique for quantitative measure-ments of bacterial cells (23). Results presented here indicatethat in logarithmic-phase derepressed cells of B. lichenifor-mis 749/C, the colloidal gold particles were present both inthe plasma membrane and the cytoplasm. The most notablecharacteristic of the distribution was the cluster formation ofthe gold-IgG particles specifically in association with theplasma membrane.

MATERIALS AND METHODSGrowth,: enzyme assay, and antibody production. B. li-

cheniformis 749/C cells were grown in low-phosphate caseinhydrolysate medium containing 0.1% glucose for 4.5 h at32°C with constant shaking (15). Alkaline phosphatase activ-ity was assayed by monitoring the release of p-nitrophenolfrom p-nitrophenyl phosphate as described previously (10);protein content was estimated by the method of Lowry et al.(16). Alkaline phosphatase was extracted and purified by apreviously described method (11). High-titer antibody wasproduced in rabbits. Four weekly intramuscular injections ofpure alkaline phosphatase (1 mg/ml) were followed by oneintraperitoneal booster injection. The serum obtained fromone rabbit was used for the isolation of IgG by ion-exchangechromatography (7). The antibody titer of the purified IgGwas determined by serial dilutions on Ouchterlony plates.

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SUBCELLULAR LOCALIZATION OF ALKALINE PHOSPHATASE 669

Preparation of colloidal gold. Colloidal gold was preparedby the method of Frens (5), with some modification. Briefly,100 ml of 0.01% HAuCl4 was heated in a flask. At the onsetof boiling, 320 p.l of 10% sodium citrate was added at once.The mixture was boiled until its color became clear wine red(10 min for a 400-ml solution). After cooling, the pH of thesolution was adjusted to 7.6 with 0.2 M K2CO3. The diame-ters of gold particles (Fig. la) show a small variation (i.e., 15to 18 nm).

Preparation of colloidal gold-IgG complex. The procedurefor the preparation of the colloidal gold-IgG complex hasbeen described by Romano and Romano (20). A purified anddialyzed aqueous solution of IgG (5 ,ug/ml) was added to theaqueous colloidal gold suspension. The mixture was shakenfor 2 min, followed by sequential addition of polyethyleneglycol (molecular weight, 20,000)-NaCl, both of which wereat a 1% final concentration. This suspension was centrifugedat 30,000 x g for 40 min, and the supernatant was discarded.The pellet was suspended in 0.1 M Tris-hydrochloride buffer(pH 7.4) containing 4% polyvinylpyrolidone (molecularweight, 10,000) and 0.2 mg of polyethylepe glycol per ml.The material was further centrifuged at 30,000 x g for 30 minto remove free IgG, and the pellet was suspended in thesame buffer. This solution contained about 109 gold-IgGparticles per ml. These particles were stable for severalmonths at 4°C. The minimal amount of IgG needed forstabilization (saturation) of the colloidal gold was deter-mined by the technique of Horisberger et al. (13) with themodification of Roth and Binder (22). A constant volume ofcolloidal gold (0.5 ml) was mixed with 50 RIl of a serialdilution of IgG. After 5 min, 0.2 ml of 10% NaCl was addedfor visual judgement of floculation, as indicated by a colorchange from red to violet or blue. Gold-IgG complex, ifprepared following the IgG-to-colloidal gold ratio as indicat-ed by the floculation test, led to poor labeling. We used a

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ratio of 100 IgG molecules to one colloidal gold particle witha 15- to 18-nm diameter. This ratio was based on equimolarconcentrations of IgG to gold. However, the surface areas ofcolloidal gold particles must be considered to arrive at aconcentration of IgG needed to saturate the surfaces ofcolloidal gold particles. In addition, polyethylene glycol wasneeded to ensure stabilization of the gold-IgG particles.

Preparation of samples. (i) Whole cells. The cells wereharvested and washed by centrifugation at 10,000 x g with0.05 M Tris buffer (pH 7.4) containing 2 mM MgCI,, 0.01 MNaCl, 100 ,ug of chloramphenicol per ml, and 50 ,ug ofsoybean trypsin inhibitor per ml, which was added toprevent protease digestion of cell-surface-bound alkalinephosphatase.

(ii) Protoplasts. The washed cell pellet was suspended at 8to 10 mg (dry weight) per ml in 0.05 M Tris-hydrochloridebuffer (pH 6.2), containing 0.65 M sucrose, 100 ,ug ofchloramphenicol per ml, 50 ,ug of soybean trypsin inhibitorper ml, 1 mM MgC92, 0.01 M NaCl, and 100 ,ug of lysozymeper ml. This suspension was incubated at 37°C for 40 minwith constant shaking. The conversion of protoplast wasmonitored by phase microscopy.

(iii) Magnesium salt extraction. Equal volumes (2 ml) ofwashed cell suspension and 2 M MgCl solution in 0.01 MTris-hydrochloride buffer (pH 7.4) were mixed and incubat-ed at 37°C for 15 min with constant shaking. This materialwas centrifuged at 30,000 x g for 30 min, and the superna-tant was separated for the assay of alkaline phosphataseactivity. The residue was washed and suspended in 0.01 MTris-hydrochloride buffer (pH 7.4).

Frozen thin sections. Both repressed and derepressed cellswere used for the preparation of frozen thinsections (8).These cells were either fixed in a mixture of O.25% glutar-aldehyde-0.4% paraformaldehyde (7) in Tris hydrochloridebuffer (pH 7.4) or in 0.5% tannic acid-0.6%-glutaraldehyde

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FIG. 1. (a) Preparation of colloidal gold used for labeling IgG; note the absence of an electron-lucid halo around the particles. (b)Preparation of gold-IgG particles; note the 2- to 3-nm electron-lucid IgG band (arrows) around these particles. Both preparations werenegatively stained with 1% ammonium molybdate. Bars, 0.1 ,um.

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670 TINGLU, GHOSH, AND GHOSH

in Iacodylate buffer (pH 7.2) (18) at 4°C for 30 min. The fixedcell suspension was washed once with appropriate buffers,followed by washing with buffers containing 30 to 50%glycerol. Portions of washed cell pellets were frozen in liquidnitrogen slush (9) as droplets and stored in liquid nitrogen.Ultrathin sections were cut at -86°C with a Sorval MT2Bmicrotome (70- to 90-nm thickness setting) with an FTSattachment (3). The sections were collected from the edge ofthe knife on a frozen droplet of 60% sucrose containing0.05% gelatin (24) formed at the end of a platinum wire. Thesections from sucrose droplets were transferred to Butvar-(Monsanto Co., St. Louis, Mo.) coated grids. The grids werewashed by floating briefly on the surface of deionizeddistilled water (0 to 40C) in a beaker to remove sucrose andwere labeled immediately.

Labeling with gold-IgG conjugate. (i) Frozen thin sections.To label and stain the frozen thin sections, the grids contain-ing the sections were treated in succession by floating ondrops, placed on dental wax, of 1% bovine serum albumin (1min), gold-IgG complex containing 109 particles per ml (10min), deionized water (3 to 5 min), and finally 1% ammoniummolybdate (2 min) for staining. All treatments were carriedout at room temperature. Treatment with bovine serumalbumin was needed for reducing surface tension and im-proving the spread of gold-IgG particles on the sectionsurface.

(ii) Protoplasts. Washed protoplasts in 0.65 M sucrosewere prefixed as described above. Afte'r prefixation, theprotoplasts were washed with 0.01 M cacodylate buffer (pH7.2) without sucrose. Protoplast suspension (1 ml) (generat-ed from 2.5 x 109 cells) was incubated with 1011 gold-IgGparticles in 100 ml of Tris-hydrochloride buffer (pH 7.4) at37°C for 30 min with constant shaking. This excess of gold-

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N4o. OF GOLD-I,G COMPLEX C108)FIG. 2. Binding of different concentrations of gold-IgG particles

with pure alkaline phosphatase (APase); addition of increasingamounts of gold-IgG particles to constant amounts of alkalinephosphatase produces increasing amounts of antigen-antibody (Ag-Ab) precipitate (ppt) based on activity of the precipitate (U) orprotein content (A). Also, interaction of gold-IgG particles withalkaline phosphatase stimulates the catalytic activity when mea-sured as a percentage of the activity present in the untreated enzyme(0). Stimulation of residual activity after the addition of lgG ischaracteristic for this enzyme.

IgG particles ensured adequate interaction of the gold labelwith the possible binding sites of cells. The gold-IgG parti-cles unbound to cell material were removed from the sam-ples by centrifugation in 10% Ficoll. The labeled sampleswere recovered as a pellet. Unbound gold-IgG particlescould not penetrate the Ficoll layer. After repeated washingin the buffer, the pellet was fixed in osmium tetroxide,dehydrated in ethanol series, and embedded in a low-viscosity resin (8). Thin sections from these resin blockswere lightly stained for 1 to 2 min with dilute Reynolds leadcitrate solution (19).

Stereology. Each photomicrograph negative (10.2 by 12.7cm) at x10,000 magnification was analyzed on a Weibelstereological table by using a line test probe containing 84line segments and 168 test points (26). Final magnification onthe screen of the counting table was x52,500. The followingdata were collected simultaneously on a multichannelcounter: (i) points on the bacterial cells or protoplasts, (ii)the number of gold particles in the cytoplasm of whole cell orprotoplast, (iii) number of gold particles in the entire areacovered by the probe, (iv) intersections of the test lines withthe inside surfaces of the bacterial membrane, and (v) thenumber of gold particles bound to the inside edge of themembrane profile. On a section profile, the interface of thecytoplasm and the inside edge of the plasma membranecould be clearly recognized at x52,500 magnification on thecounter screen. There was rarely any problem distinguishingthe gold particles bound to this interface. The described dataabove were used to compute (i) the number of gold particlesdistributed on the unit area of cell sections [N(C)A7], as wellas on the unit area surrounding the cells representing non-specific background gold particles; (ii) the number of goldparticles bound to unit length of the membrane [N(A)BT]. Thefollowing relationships were used for the computation (27):

2 . T(c)N(C)AT = v . 2 (1)

where T(c) is the total number of gold particles bound to thecell, d is the short test line length, and P1c1 is the totalnumber of test point on the cells.

ThMlN(M)B x KIi.P(C) (2)

where ThM) is the total number of gold particles bound to themembrane profile, Ii is the total number of intersections oftest line with the cells, and K is a constant equal to(41 * Tr * d) x total number of test lines.

RESULTSSpecificity of antibody and nature of conjugation of IgG

with colloidal gold. The purified IgG used in this experimentwas specific for alkaline phosphatase because the antibodywas raised against highly purified enzyme. Furthermore, theOuchterlony double diffusion technique showed a clear bandwith pure alkaline phosphatase and MgClI extract of dere-pressed cells; however, no band was formed when theextract of repressed cells was used. This specificity of IgGwas retained after forming a complex with gold particlessince the colloidal gold-IgG comple'x precipitated pure alka-line phosphatase (Fig. 2). This antigen-antibody interactionstimulated the catalytic activity of the enzyme. Such stimu-lation has been shown to be a characteristic of alkalinephosphatase isolated from B. licheniformis (A. Ghosh, un-

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published data). The results in Fig. 2 show that increasingamounts of pure alkaline phosphatase could be precipitatedwith gold-IgG particles if increasing numbers of such parti-cles were added to constant amounts of pure alkaline phos-phatase. On addition of a sufficiently high number of parti-cles, saturation with the enzyme described above could bedemonstrated. Thus, the probe is sensitive to variations inthe number of antigenic sites present in the frozen thinsections of the organism.Aggregation of immuno-probes (e.g., gold-IgG complex or

ferritin-IgG conjugate) generates artifacts of labeling. Asingle antigenic site may appear as a cluster if labeled withaggregated probes. Comparison of Fig. la and b shows thelack of aggregation of colloidal gold particles in a suspensionbefore and after complex formation with IgG. A thin elec-tron-lucid band of uniform thickness (2 to 3 nm) shows IgGcoating on the gold-IgG particles (arrows, Fig. lb). Theresults shown in Fig. 2 also indicate that the IgG present onthe gold-IgG particles could be saturated with purifiedantigen (i.e., alkaline phosphatase).

Specificity of labeling. The results presented here originat-ed from both frozen thin sections and conventional thinsections of resin-embedded cells. Hence, some characteris-tics of frozen thin sections are discussed here which will helpin interpretation of the results.

Interpretation of frozen thin section images is differentfrom the interpretation of thin sections obtained from con-ventionally embedded specimens. Unlike resin-embeddedcells, frozen cells are embedded in amorphous ice; hence,the cell structure and its external matrix are not supportedby a continuum of polymer. Both mechanical stress and aminute amount of heat generated during advance of the knifemay cause small movements of the ice-embedded cells. Suchminor dislocation of cells during cutting frequently generatedprofiles of sections which appeared to be partially cross cutand partially cut in ad oblique direction (Fig. 3a through d).In Fig. 3c is shown a distinct cell membrane and cell wallprofile of the section, but a portion of the section edgeappears fuzzy, because a slight shifting of the cells during theadvance of the knife resulted in an oblique cut of theremaining part of the cell. Hence, asymmetry of the sectionedge is a common feature of the frozen thin section. Thefrozen thin sections were stained with ammonium molyb-date, which generated a contrast based on the principles ofnegative staining. Therefore, multiple layering of the mem-brane or wall could not be seen; the membrane profileappeared as a single electron-lucid layer (Fig. 3c). As themembrane is electron lucid and the colloidal gold is electrondense, the binding of gold-IgG particles to the membranecould be distinctly seen in labeled sections at x52,500magnification. Inspection of Figs. 3a through c clearly showsthis binding of gold-IgG particles to the membrane (arrowFig. 3c).

Specificity of binding was confirmed by the labeling of (i)frozen thin sections pretreated with anti-alkaline phospha-tase antibody to block the antigenic sites, (ii) frozen thinsections of cells after magnesium salt extraction to removecell-bound alkaline phosphatase, and (iii) frozen thin sec-tions of repressed cells.

Labeling of derepressed cells is seen in Fig. 4a. Bothclusters of gold-IgG particles (arrows, Fig. 4a) and singlegold-IgG particles (arrowheads, Fig. 4a) are distributed inthe sections. To block the alkaline phosphatase antigenicsites, the sections were pretreated with different concentra-tions of pure IgG, followed by labeling with gold-IgG parti-cles. Frozen thin sections pretreated with 600 ,ug of IgG per

ml of incubation mixture rarely bound any gold-IgG particles(Fig. 4b). Magnesium salt-extracted samples, which retainedonly 15 to 20% of the cell-bound enzyme, showed thebinding of only a few gold-IgG particles (Fig. 4c). Finally,the repressed cells also showed almost no binding (Fig. 4d).These qualitative results were confirmed by quantitativeestimations, which will be described.The following criteria justify the application of the stereo-

logical technique to quantitate the binding of antibody-labeled colloidal gold. (i) The labeled material was applied tothe section surface; therefore, all of the exposed antigenicsites were available for labeling. (ii) The label was specifical-ly bound to the section surface because the distribution ofgold-IgG particles bound to the background was much lowerthan the binding with the cellular material. (iii) The labelingdid not require diffusion through the membrane; hence, novariability could arise from differences in the cytoplasmicdensity or any other hindrance to diffusion. (iv) The thick-ness of the sections did not influence the quantitationbecause only the section surface was labeled. (v) Serialsections from any one bacterium did not predominate in thelabeled sections collected on any one electron microscopicgrid because frozen thin sections did not form ribbons.Therefore, micrographs for quantitative analysis obtainedfrom these grids maintained randomness in representing thebacterial population.The number of gold-IgG particles bound per square mi-

crometer of section surface was estimated (Table 1). Pre-treatment with 600 p,g of IgG per ml caused 82% reduction ofgold-IgG particle binding, suggesting 82% blocking of anti-genic sites. Magnesium chloride extraction caused 60%reduction of gold-IgG particle binding, suggesting 60% re-moval of alkaline phosphatase, which is consistent withbiochemical assays of enzyme content after MgCI2 extrac-tion. Repressed cells showed 93% lower binding comparedwith derepressed cells. Washed protoplast suspensionsshowed 46% reduction of binding; biochemical estimationshowed that almost 50% of cell-bound enzyme was lostduring protoplast formation. The results in Fig. 5 show thatas the concentrations of IgG pretreatment were increased,more antigenic sites were blocked; finally, at a concentrationof 600 ,ug/,ul, there was very little binding with the sectionsurface, suggesting total blockage of the alkaline phospha-tase antigenic sites. Gold-IgG particles fully neutralized withpure alkaline phosphatase before labeling did not significant-ly label the frozen thin sections.

Distribution. The distribution of gold-IgG particles bound

TABLE 1. Determination of the specificity of gold-IgG labeling ofB. licheniformis 749/C frozen thin sections"

No. of goldparticles

Sample per ,um-surfacearea

Derepressed cells .................................. 12 ± 1.2"Derepressed cells pretreated with anti-alkalinephosphatase IgG (260,ug/ml) ......... ............. 3 ± 0.3

Derepressed cells after MgCI2 extraction.............. 3 ± 0.3Repressed cells .................................... 1 ± 0.1Protoplasts .................................... 6 ± 0.6Protoplasts pretreated with anti-alkalinephosphatase IgG (260 >±gIml) ......... ............. 2 + 0.2

" Electron micrographs of labeled frozen thin sections were used forstereological analysis as described in the text.

b Values are means ± standard error.

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to the membrane and cytoplasm was studied in frozen thinsections of whole cells and conventional resin-embeddedthin sections of prelabeled protoplasts. In frozen thin sec-tions of whole cells, the gold-IgG particles were bound to the

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plasma membrane, the cell wall, and the cytoplasm. Thoseparticles bound to the membrane were present mostly inclusters; however, a significant number of single particleswere also present. The clusters may be small, consisting of

FIG. 3. Characteristics of labeling of cell envelope in frozen thin sections. Sections were prepared from tannic acid-glutaraldehyde-fixedcells and stained with 1% ammonium molybdate. (a) Envelope-associated particles (box) are bound to inner and outer membrane surfaces. (band c) Comparison of these two micrographs shows that envelope-associated particles may form small (arrowheads) or large (arrow) clusters.(d) More particles are associated with the cytoplasm than with the cell envelope. Bars. 0.2 tim.

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F16. 4. Frozen thin sections labeled with gold-IgG particles; these preparations were used for the quantitative estimation of gold-IgGparticle distribution in association with the cell envelope or cytoplasm. (a) Derepressed cells. Note the binding of gold-IgG particles inclusters, with the cell envelopes (arrows) and isolated individual particles in the cytoplasm (arrowheads). (b) Derepressed cells labeled afterblocking the antigenic sites with 600 p.g of anti-alkaline phosphatase IgG per ml. (c) Derepressed cells labeled after extraction of alkalinephosphatase with 1 M MgClI. (d) Labeled repressed cell. Bars, 0.5 ,um.

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CONCENTRATION OF IgG (Qg/ml)FIG. 5. Effect of blocking the alkaline phosphatase antigenic

sites of the frozen thin sections by treatment with different concen-

trations of anti-alkaline phosphatase antibody. The number of goldlabels bound per square micrometer of section surface were countedafter IgG pretreatment; 600 ,ug of IgG per ml almost totally blockedthe antigenic sites of thin sections.

three or four gold-IgG particles (box, Fig. 3a; arrowheads,Fig. 3b), or large, consisting of 10 or more particles (arrow,Fig. 3c). It should be noted that the size' of the immuno-probe used in this study was 15 to 18 nm; therefore,differences in the sites of binding of a single particle within15 to 18 nm is insignificant as far as the location of antigenicsites. However, presence of the clusters strongly suggeststhe presence of groups of antigenic sites within a structure.Frequently, these clusters span the membrane, periplasm,and cell wall (Fig. 3a and c), suggesting the spread ofantigenic sites through a specific envelope region. Inspectionof a large number of sections suggested that more gold-IgGparticles were bound to the cytoplasm than to the membrane(Fig. 3d). The relative distribution of gold-IgG particlesbetween the membrane and the cytoplasm was determinedby stereological analysis. There were, on the average, 12gold-IgG particles bound per jxm2 of section surface. Asection surface of 1 ,um2 is delimited by a 1.5-pum membraneprofile, which contained, on the average, three gold-IgGparticles. Therefore, 27% of the gold-IgG particles werebound to the plasma membrane and 73% were bound to thecytoplasm. In a large number of experiments, a range of 25to 35% binding was observed in the membrane and 65 to 75%was observed in the cytoplasm. This distribution in bindingto the cytoplasm relative to the membrane is much higherthan the amount determined by fractionation of rupturedcells (8). It is possible that a substantial amount of cytoplas-mic enzyme was nonspecifically relocated during cell frac-tionation to the periplasm or cell walls. However, thecytoplasmic alkaline phosphatase may be immuno-reactivebut catalytically inert, as was observed for penicillinase inthis organism (7).

Characteristics of the labeling of plasma membrane werefurther studied by using mildly prefixed osmotically shockedprotoplasts. This preparation prevented large-scale releaseof alkaline phosphatase, and the interior of the protoplastbecame available for labeling. In a low-magnification micro-graph (Fig. 6a), gold particles are seen to be primarily bound

to the plasma membrane. These are present both as clusters(arrow, Fig. 6a) and as single particles (arrowheads, Fig. 6a).The binding of gold-IgG particles on both sides of the plasmamembrane is clearly seen in Fig. 6b; such a large amount ofbinding in a single profile was not common, but this is a clearexample which demonstrates that enzyme molecules existon both surfaces of the plasma membrane; similar labelingwas also seen in frozen thin sections. In high-magnificationmicrographs (Fig. 6c and d), labels are seen to be bound inclusters to both inner (Fig. 6c) and outer (Fig. 6d) parts of theplasma membrane. Frequently, labels appear to span themembrane thickness (arrowhead, Fig. 6d). Low binding withthe cytoplasmic material in these peeparations resulted fromrestricted movement of the gold-IgG particles through cyto-plasmic material cross-linked with aldehyde.

DISCUSSIONIn vivo high-resolution localization of secretory proteins is

crucial for the understanding of secretory mechanisms ofproteins at the molecular level. In most cases, subcellularlocations of these proteins are determined in cell fractionsobtained from ruptured cells by differential or density gradi-ent centrifugation (17; Ghosh and Ghosh, in press). Howev-er, during cell fragmentation and subcellular fractionation,proteins may be relocated to structures which are differentfrom the in situ location of the proteins (17). Therefore, smallchanges in the location of secretory proteins at the molecularlevel during secretion cannot be detected. In specimensprepared for the subcellular distribution study by cytochem-istry or immuno-cytochemistry, these relocation artifacts arelow, because cellular organization remains largely intact inthese preparations. In a previous report, it has been shownby ultrastructural cytochemistry that alkaline phosphatase,which is a secretory protein in B. licheniformis 749/C, ismainly bound to the inside surface of the plasma membrane,and a small amount is bound in the cytoplasm (8). Thecytochemical technique depends on the catalytic activity ofthe enzyme molecules for their localization reaction, where-as by the immunological technique, proteins are detected byspecific features of the structures of molecules. To use theimmuno-electron microscopic technique to obtain quantita-tive data and to assess pattern formation by protein mole-cules, a number of criteria must be met. This techniquerequires labeling of the specific antibody with an electron-dense marker. The use of colloidal gold as a marker hasseveral advantages: (i) unlike ferritin, colloidal gold does notpolymerize, (ii) gold-IgG particles can be prepared by simplemanipulations and, unlike ferritin, do not require cross-linking with a bifunctional reagent, and (iii) colloidal gold ofmany different sizes can be prepared by simple procedures.The pitfall of using colloidal gold as a label arises from thehigh electronegative surfaces of gold particles. Unless theseelectronegative particle surfaces are fully saturated by bind-ing with a protein or any other appropriate material, theparticles may nonspecifically associate with the labeledsamples. The protocol for preparing saturated colloidal goldparticles should be rigidly followed; otherwise, the labelingwill be highly nonspecific.

Previous reports have indicated that fixed and embeddedthin sections are used for gold labeling (2, 21); in manypreparations, however, the chances of denaturation and lossof antigenicity are high. In fact, the antigenicity of B.licheniformis alkaline phosphatase was mostly lost afterfixation and embedding in conventional epoxy resin. There-fore, in the present study, we used primary labeling of mildly

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FIG. 6. Prefixed and osmotically shocked protoplasts were labeled with gold-lgG particles and then embedded and sectioned. (a) Note thatthe membrane is labeled either with a cluster (arrow) or with single particles (arrowheads); however, due to the lack of flow of labeled gold-IgG particles in these cells prefixed with aldehyde, the cytoplasm was rarely labeled. (b) Distribution of label can be seen on both sides of theplasma membrane. (c and d) High-magnification micrographs show clear binding of gold label in clusters at the plasma membrane inner andouter surface, and one gold particle is seen partially overlapping the membrane bilayer (arrowhead; d). Bars, 0.1 p.m.

fixed frozen thin sections. Prefixation prevented gross dislo-cation of protein molecules caused by mechanical stressduring sectioning or by molecular reorganization duringthawing of frozen sections required for labeling. Damage ofthe cells due to ice-crystal growth during freezing wasminimized by washing with 30 to 50% glycerol and by rapidfreezing. We have shown that this method of freezing causesminimal damage to the B. licheniformis structure (9). Dam-age to the frozen sections during thawing was prevented bysucrose, which was the substrate for the collection ofsections (24). After labeling, the frozen thin sections werewashed and stained with ammonium molybdate. This short

treatment contrasts with previous methods of refixation,staining, and embedding in Methyl Cellosolve (Union Car-bide Corp.) after labeling (24); such treatment may causeappreciable dissociation and relocation of gold labels insmall bacterial cells.

In spite of the precautions mentioned above, cell damagecould not be fully prevented, e.g., compression by anextracellular ice crystal caused narrowing of some cells (Fig.3a), and after thawing, small ice crystals occasionally gener-ated bubbles in the cytoplasm (Fig. 3d and 4a). This distor-tion of cells did not appreciably affect the data on thesubcellular distribution and cluster formation of gold-IgG

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particles bound to the plasma membrane. The damage didnot affect all cells uniformly. Gross changes could only beseen in a few cells. Therefore, samples prepared frommultiple experiments were examined. The results of ourstudy are not only based on a few chosen micrographs; datagenerated from the cell population at random provided thebasic guideline for presenting micrographs included here.Conclusions on the nature of membrane labeling are basedon corroborating data from the application of differenttechniques of specimen preparation. One should, however,be aware that the application of the immuno-labeling mnethodpresented in this paper certainly requires extensive prelimi-nary evaluation in another system because highly complex,poorly understood factors influence the effect of freezing,thawing, and immuno-labeling.The most notable result of this study is that membrane-

bound gold-IgG particles were present in clusters. However,such grouping of gold-IgG particles was infrequent in thecytoplasm, although a few could be seen. The possibility thatcluster formation may occur nonspecifically as an artifact isremote. Before the addition to cell samples, the gold-IgGparticles were unaggregated and uniformly dispersed. Clus-ters of gold-IgG particles were seen only for alkaline phos-phatase, whereas labeling for penicillinase was dispersed (G.Tinglu, A. Ghosh, and B. K. Ghosh, Abstr. EMSA 1983, no.614). These membrane-bound clusters, when examined un-der high magnification, showed that individual gold particleswere clearly separated from each other; in nonspecificaggregates, however, individual particles remained in closecontact with each other. If the cells devoid of alkalinephosphatase were treated with gold-IgG particles or theantigenic sites of the cells were blocked by specific antibodybefore labeling, clusters were not formed. This alkalinephosphatase-specific cluster of gold-IgG particles in associa-tion with the plasma membrane indicates the presence of agroup of alkaline phosphatase molecules bound to specificsites in the plasma membrane.We interpret these immunoelectron microscopic data as

being suggestive of the existence of specific alkaline phos-phatase binding sites (receptors) in the plasma membrane ofB. lihheniformis 749/C. The bacterial cells may be compara-ble to animal cells in that they have a membrane receptor-mediated secretory phenomenon (17; Ghosh and Ghosh, inpress). Clear experimental data, however, are still lacking.Ongoing experiments are in progress in our laboratory toisolate this alkaline phosphatase receptor from B. lic henijor-mis. A recent immunoelectron microscopy experiment hasshown that in an alkaline phosphatase secretion-blockedmutant of B. licheniformis 749/C/NM105 (15), the mem-brane-bound gold-IgG particles were low, but cytoplasm-bound particles were abundant. It is possible that the ab-sence of a putative secretion receptor for alkalinephosphatase blocked the secretion of this enzyme in themutant.

ACKNOWLEDGMENTS

This study was supported by National Science Foundation grantsPCM 6110613 and 8108665 and by Department of Energy contractDE-AS05-80 ER10703.We express our appreciation to Marian Glenn and Dean Handley

for their valuable suggestions. Gloria Trechock provided expert helpin preparing this manuscript.

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Documents

Subcellular Localization of Alkaline Phosphatase in Bacillus