Visualization of the intact endoplasmic reticulum by

immunofluorescence with antibodies to the major ER glycoprotein,

endoplasmin

G. L. E. KOCH, D. R. J. MACER and M. J. SMITH

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England

Summary

Antibodies to endoplasmin were used to examinethe morphology of the endoplasmic reticulum(ER) by immunofluorescence on permeabilizedplasmacytoma and fibroblastoid cells. In unfixedcells, permeabilization led to a pronounced ves-iculation of the ER. Therefore cells were firstfixed lightly prior to permeabilization with deter-gent. Fibroblastoid cells gave a characteristicreticular pattern surrounding the nucleus withclear staining of the nuclear membrane. Plasma-cytoma cells, in the conventional fluorescencemicroscope, gave a cisternae-like pattern. Opticalsectioning with a confocal scanning lasermicroscope gave a distinct pattern of con-centric cisternae similar to those obtained with

transmission electron microscopy on cell sec-tions. The overall morphology of the ER in suchcells could be revealed by serial optical section-ing. Evidence was obtained that the ER does notundergo extensive vesiculation during mitosis inplasmacytoma cells. Using anti-endoplasmin im-munofluorescence monitoring, conditions weredeveloped for the retention of ER morphology inunfixed, permeabilized cells.

These studies illustrate the value of endoplas-min as a general marker for the analysis of ERmorphology in different types of cells by immu-nofluorescence microscopy.

Key words: endoplasmic reticulum, ER visualization, ERimmunofluorescence, endoplasmin antibodies.

Introduction

The endoplasmic reticulum (ER) consists of a complexset of membrane lamellae and tubules, which is the siteat which many major biosynthetic processes such asprotein secretion, membrane assembly and organellebiosynthesis are initiated (Porter et al. 1945; Palade &Porter, 1954; Porter, 1953; Krstic, 1979; Alberts et al.1983). Although there have been striking advances inthe understanding of the biochemistry of these pro-cesses, several fundamental questions remain about therelationship between ER structure and function. Theseinclude: (1) the possibility of segmentation beyond themorphologically distinct rough and smooth ER; (2) thedistribution of resident luminal as well as secretoryproteins within the ER; (3) identification of the sitesfrom which material, presumably in a vesicular form,leaves the ER; (4) the fate of the ER itself during celldivision and other perturbations. Examination of manyof these questions remains difficult because they re-quire visualization of the entire organelle in situ, rather

Journal of Cell Science 87, 535-542 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

than parts of it as is possible by electron microscopy ofthin sections.

As pointed out previously (Terasaki et al. 1984), theoriginal whole-mount electron micrographs of Porterand co-workers (see above) remain among the bestexamples of ER visualization in situ. Since then, themost significant advance has probably been the studiesof Terasaki et al. (1984), which have shown that theentire ER can be rendered visible by fluorescencemicroscopy with certain cationic dyes that appear topartition preferentially into the ER. However, thesereagents are not completely specific for ER and can leadto artefactual staining of other membrane organelles.

Louvard et al. (1982) have shown that it is possible touse antibodies against putative ER-specific antigens tovisualize the ER by immunofluorescence microscopy.However, it is not possible to assess the generalapplicability of the approach used, since it employed apolyspecific antibody, directed against a large numberof undefined protein antigens, which was dependent on

535

extensive absorption to render it specific towards theER.

A general approach to the visualization of the ER byimmunofluorescence microscopy should employmarkers that have been formally demonstrated to bespecifically located in the ER, are known to be presentin a wide variety of cell types, are sufficiently abundantto facilitate access in relatively intact cells and towardswhich strong monospecific antibodies can be produced.Recently it was shown that the ER of all vertebrate cellscontains a major glycoprotein that is undetectable inany other organelle and towards which it is possible toobtain monospecific antibodies for immunolocalization(Koch et al. 1986). The glycoprotein, which is calledendoplasmin, therefore satisfies all the criteria men-tioned above as a general marker for the visualization ofER in cells by immunofluorescence microscopy.

In this study, the suitability of endoplasmin as an ERmarker in immunofluorescence microscopy on bothflattened as well as large, rounded cells such as plasmacells was examined. The results indicate that endoplas-min is indeed suitable for this purpose, particularlywhen used in combination with a newly developedconfocal scanning microscope. The morphology of theER has been characterized under a variety of conditionsby this approach, and conditions developed for theretention of ER morphology in a 'cell-free' system.

Materials and methods

CellsAll cells were grown in RPM1 1640 medium (Gibco) with10 % foetal calf serum, 100 units per ml penicillin/streptomy-cin and 4mM-L-glutamine. NIH-3T3 cells were grown ontissue-culture plastic substrate and harvested with tryp-sin/EDTA (Todaro & Green, 1963). MOPC-315 plasma-cytoma cells were grown in suspension.

NIH-3T3 cells were prepared for microscopy by placing adroplet of a 1X107 cells ml"1 suspension in growth mediumon a clean glass slide. After 30min at 37°C for attachment,slides were incubated in growth medium at 37"C to permitspreading to occur. This was usually complete in about 2h.

MOPC-315 cells were suspended in phosphate-bufferedsaline (PBS) (Koch & Smith, 1986) and a droplet was placedon a polylysine-coated clean glass slide. Cells became at-tached in about IS min.

Permeabilizing of cells for immunofluorescencelabellingUnfixed cells were treated with O'Ol % saponin in PBS for2 min and then fixed with 3-5% formaldehyde in PBSfor 15 min.

Cells were prefixed with 3-5% formaldehyde in PBS for15 min.

Final permeabilization was effected with 0-2% saponin inPBS for 15 min.

Antibodies and lectinAffinity-purified antibodies to endoplasmin were prepared asdescribed previously (Kochef al. 1986). Fluorescein-labelledgoat anti-rabbit immunoglobulin (Ig) was purchased fromSigma and used at 1:20 dilution.

Fluorescein-labelled wheat-germ agglutinium and con-canavalin A (ConA) were purchased from Sigma.

Immunofluorescence labellingCells that had been treated with 0 2 % saponin (see above)after fixing were treated with antibody for 15 min at roomtemperature, washed extensively with 0-2% saponin/PBSand developed with fluorescent second antibody for 15 min atroom temperature. After extensive washing, samples weremounted in 90% glycerol with 1 % phenylene diamine, andsealed with varnish. Controls were carried out without thefirst antibody layer. Staining with fluorescent wheat-germagglutinin was as above.

Immunofluorescence microscopySamples were viewed in a Zeiss Universal epifluorescencemicroscope equipped with a narrow band fluorescein exci-tation filter. Photographs were taken with Kodak Tri-X film.

Confocal laser scanning fluorescence microscopyThis was carried out on an instrument (the MRC systemconfocal fluorescence microscope) designed by Dr J. White,details of which will be described elsewhere (White, Amos &Fordham, unpublished). The instrument employs a scanningbeam of laser light and confocal optics to illuminate andanalyse relatively thin regions of the specimen. This providesconsiderable improvements in resolution and permits serialoptical sectioning.

Results

ER vesiculation in permeabilized cellsPrevious studies have shown that cells treated with thedetergent saponin became depleted of their cytoplas-mic contents but retain all the endoplasmin, suggestingthat the ER is still intact (Koch et al. 1986). Such'shells' could be ideal for analysis of the ER. However,immunofluorescence studies with anti-endoplasmin(Fig. 1) show that although there is considerableintracellular staining this is located in vesicular struc-tures, suggesting that significant distortion of the ERhas occurred upon permeabilization. Electron mi-croscopy of the shells (Fig. 1) confirms that the ER,identified by the attached ribosomal particles, hasindeed dilated and vesiculated in the permeabilizedcells. Thus the permeabilized cells do not appearsuitable for analysis of ER morphology.

Anti-endoplasmin staining of fixed, permeabilizedcells

To overcome the distortion caused by direct per-meabilization of cells, a light-fixation step was intro-duced. It was found that fixation with formaldehyde

536 G. L. E. Koch et al.

Fig. 1. Immunofluorescence with anti-endoplasmin on unfixed cells permeabilized with saponin. A. 3T3 cells were treatedwith O'Ol % saponin/PBS, fixed with formaldehyde and stained with anti-endoplasmin/FITC-goat anti-rabbit Ig asdescribed in Materials and methods. Note the vesicular pattern of staining. Bar, S jlm. B. Cells prepared as above wereprepared for electron microscopy as described in Materials and methods. Note the dilation of ribosome-lined rough ER andthe apparent dispersal of the cisternae of the Golgi apparatus. Bar,

yielded samples that possessed the correct balance ofstability and permeability to permit access of antibodiesto the interior of the ER. In such preparations, anti-endoplasmin gave a characteristic reticular patternaround the nucleus, with clear staining of the nuclearmembrane (Fig. 2). The latter is characteristic ofendoplasmic reticulum (Krstic, 1979), and is a usefulindicator of the permeabilization procedure.

The staining pattern with anti-endoplasmin is clearlydifferent from that obtained with the Golgi-indicatorwheat-germ agglutinin (Fig. 2), which gives a tightpolar cap at one pole of the nucleus and no staining ofthe nuclear membrane.

Although pre-fixation was also useful for the examin-ation of round cells such as plasmacytoma cells, whichyielded a pattern suggesting an intracellular membra-nous reticulum (Fig. 3), the thickness of the cellsinterfered with the visualization of the overall patternof fluorescence. However, examination of the samepreparation by confocal laser scanning fluorescencemicroscopy (Fig. 3C) revealed the striking concentricpattern characteristic of the ER in plasma cells. Theresolution of the technique was sufficient to permit theseparation of individual cisternae. Because the optics ofthe system permit relatively thin (0-7/Urn) sections tobe examined, there is a dramatic increase in signal to

ER visualization by immunofluorescence 537

noise above that obtained with the conventional micro-scope. This also permits the examination of the generalmorphology of a complex organelle like ER by serialoptical sectioning microscopy (Fig. 4). Although theanalysis is not complete, it illustrates the feasibility ofusing this approach for the visualization of ER mor-phology by immunofluorescence microscopy usingantibodies to an ER marker such as endoplasmin.

Serial optical sectioning of stained plasmacytomacells, which were identified as being in mid-division by

Fig. 2. Immunofluorescence with anti-endoplasmin onfixed cells permeabilized with saponin. A,B. 3T3 cells werefixed with formaldehyde, permeabilized with 0 2 %saponin/PBS and stained with anti-endoplasmin asdescribed in Materials and methods. C. Cells prepared asabove stained with FITC-wheat-germ agglutinin. Note theperinuclear cap typical of the Golgi apparatus. Bar, 5;um.

their general morpholog}' and the absence of nucleus,revealed an apparently continuous structure with nosigns of extensive vesiculation (Fig. 5).

Preservation of ER morphology in permeabilized cellsThe dilation and vesiculation of the ER that occurswhen cells are permeabilized with saponin (Fig. 1)

Fig. 3. Immunofluorescence with anti-endoplasmin onplasmacytoma cells. MOPC-315 cells were fixed withformaldehyde followed by permeabilizing with 0-2%saponin/PBS. Labelling was with anti-endoplasmin andFITC-anti-rabbit Ig as described in Materials andmethods. A,B. The same field of cells examined by phase-contrast and epifluorescence, respectively. Bar, 10 Jim.C. The same sample examined by confocal laser scanningfluorescence microscopy. Magnification was not measureddirectly for samples analysed by this technique.

538 G. L. E. Koch et al.

suggested that osmotic effects might be involved.Therefore permeabilization with saponin was carriedout in the presence of a relatively impermeant sugar,sucrose, to overcome these putative osmotic effects.The results (Fig. 6) show that there is indeed a

Fig. 4. Serial optical sectioning of the endoplasmicreticulum by confocal laser scanning fluorescencemicroscopy. The field shown in Fig. 3C was sectionedoptically at various levels from top to bottom. Each sectionis about 0'7/im thick.

significant improvement, in that the ER in such cellsretains the morphology observed with pre-fixed cells. Aparticular advantage of this procedure is that it im-proves the general accessibility of the ER to antibodiesand thereby provides improved visualization by immu-nofluorescence.

Comparison between anti-endoplasmin and otherfluorescent probes for ER membranesA comparison was carried out between anti-endoplas-min, cyanine dyes (Terasaki et al. 1984) and fluor-escent ConA for the visualization of the ER in plasma-cytoma cells (Fig. 7). Although the latter do reveal theER, they are clearly less specific, both also reactingwith the plasma membrane and probably with othernon-ER membranes. Therefore, although useful re-agents, cyanine dyes and fluorescent ConA are of lessvalue than anti-endoplasmin for the specific visualiza-tion of the ER. Fig. 7 also shows examples of cells thatwere double-labelled with anti-endoplasmin and anti-tubulin. Cells that are clearly mitotic, as shown by thepresence of well-developed spindles, show essentiallyintact ER, confirming the above-mentioned studies(Fig. 5), which showed no evidence for gross ves-iculation in mitotic cells.

Discussion

These studies have shown that antibodies to the majorendoplasmic reticulum glycoprotein, endoplasmin, canbe used to visualize this organelle in its entirety byimmunofluorescence microscopy. In the standard pro-tocol, cells were lightly fixed with formaldehyde toretain the integrity of the structure and then per-meabilized with detergent. Remarkably, even undersuch circumstances access of the antibodies to endo-plasmin, which is probably a luminal protein of the ER(unpublished data), is not precluded and a strongfluorescence signal is obtained. This is probably areflection of the fact that endoplasmin is a veryabundant luminal protein. Therefore even the limitedpermeability achieved by treating fixed cells with amild detergent such as saponin seems sufficient.

One of the objects of developing a procedure forvisualizing the ER in its entirety was to monitor ERmorphology in permeabilized cells. It is clear that evengentle treatment of cells with saponin, which generatespreparations with a relatively intact ER since theendoplasmin is fully retained (KocheZ al. 1986), causesconsiderable distension and some vesiculation. Suchpreparations are inadequate for the study of the ERstructure and function, since the organelle is consider-ably distorted. However, use of an osmotic stabilizersuch as sucrose overcomes this problem and generateswell-permeabilized cells without vesiculation of theER. We expect that such preparations will prove

£7? visualization by immunofluorescence 539

Fig. 5. Morphology of the endoplasmic reticulum in MOPC-315 mitotic cells. Three sections at differing levels (top tobottom) are shown for each mitotic cell. Note the appearance of an intact reticulum in both cells.

particularly useful cell-free systems for the analysis ofthe ER.

The amenability of the intact ER to visualizationwith anti-endoplasmin has permitted the examinationof a concept that has intrigued cell biologists over a longperiod, i.e. what is the fate of the ER during celldivision? The first direct attempt to examine the issuein HeLa cells (Robbins & Gonatas, 1964) indicatedthat the ER vesiculates and fragments away from beinga single continuous structure in mitotic cells. This hassubsequently been confirmed in other types of cells (seeZeligs & Wollman, 1979, and references therein),although there have been sporadic reports to thecontrary (Melmed et al. 1973; Redman & Sreebny,1970; Pictet et al. 1972; Jimbow et al. 1975). A majorlimitation of these studies is that they have examined arelatively few isolated thin sections and extrapolated tothe overall morphology of the ER. The validity of suchan extrapolation is highly questionable for such acomplex organelle. Furthermore, no previous study

has addressed the question of the continuity or other-wise of the ER across the contractile ring. In this studyit was possible to examine the ER right across dividingcells, including the region of the contractile ring, anddemonstrate the apparent continuity of the organelleacross this region. It is not yet clear whether theseobservations are unique to cells such as plasmacytomacells, which contain a large amount of ER, but furtheranalyses on other types of cells with antibodiesto endoplasmin should help to clarify the generalquestion.

One of our major objectives during these studies hasbeen to obtain preparations of endoplasmic reticulumthat are accessible to external reagents, but whichretain the normal morphology of the ER in the cell.Fig. 7 shows that such preparations can be obtainedprovided suitable osmotic stabilizers are included dur-ing the permeabilization of the cells. Although exten-sive studies have been carried out on ER function withmicrosomal membrane preparations (De Pierre &

540 G. L. E. Koch et al.

Fig. 6. Stabilization of endoplasmic reticulum in permeabilized unfixed cells. MOPC-315 cells were equilibrated with 9%sucrose/PBS at 4°C. Saponin was added to 0 0 1 % and, after 15min at 4°C, cells were developed with anti-endoplasmin asdescribed in Materials and methods, and examined with the confocal laser scanning microscope. Note the reduction in ERvesiculation compared with that in Fig. 1.

Fig. 7. Immunofluorescence labelling with different reagents for ER. Cells were prepared as described in Materials andmethods, and stained with: A, D10C6 (Terasaki et al. 1984); B, FITC-Con A (Sigma, dil. 1:20); C, anti-endoplasmin( + FITC goat anti-rabbit Ig); D, anti-endoplasmin and anti-tubulin (see Koch et al. 1986).

ER visualization by immunofluorescence 541

Dallner, 1975), these are highly disorganized replicasof the in situ organelle and not suitable for detailedanalyses of the relationship between ER structure andfunction. In contrast, it is expected that the above-mentioned preparations from the plasmacytoma cell,which retain the essential morphology of the ER butwhich are accessible to external manipulation, willprovide a more realistic model for the analysis of ERfunction in a cell-free system.

We thank Dr J. White for performing the microscopy withthe MRC system confocal fluorescence microscope, and DrW. B. Amos for helpful discussions.

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(Received 5 December 1986 - Accepted 10 February! 1987)

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