TISSUE & CELL 1978 10 (2) 365-388 Published by Longman Group Ltd. Printed in Great Britain

G. L. BROWN and M. LOCKE

NUCLEOPROTEIN LOCALIZATION BISMUTH STAINING

BY

ABSTRACT. Experiments on isolated mouse liver nuclei involving enzyme digestion, the crosslinking of amino groups and alkaline hydrolysis demonstrate that bismuth binds to nucleoproteins through amino and phosphate groups. Analysis of the nucleoproteins extracted with salt and acid solutions in conjunction with bismuth staining after these treatments suggests that: (1) a bismuth amino group interaction occurs on ribonucleo-protein particles, histones and perhaps some non-histone chromosomal proteins, and (2) bismuth phosphate binding is specific for one, or all, of three distinct species of non-histone proteins. These results suggest that histones not tightly bound to DNA through their amino groups are present on interchromatin granules, the presumed transcriptionally active regions of chromatin. Phosphorylated non-histone proteins are also localized at these sites. Staining with heavy metals such as bismuth may be the best method for high resolution localization of nucleoproteins involved with regulating gene activity and maintaining chromatin structure.

Introduction

amines on histones would visually establish the areas of chromatin involved with gene transcription as well as confirm the roles of these classes of proteins in controlling chromatin structure and activity. The development of bismuth as a stain for electron microscopy that can localize amine, amidine and phosphate groups (Locke and Huie, 1975, 1976a, b, 1977) suggested that it could be signally useful for the localization of chromatin-associated proteins. Bismuth staining of sections is relatively non-specific and has been used to show ferritin (Ainsworth and Karnovsky, 1972) glycogen, lysosomes, ribosomes and aldehyde groups resulting from periodic acid oxidation (Ainsworth et al., 1972). Bismuth also intensifies uranyl and lead staining (Riva, 1974; Barret et al., 1975) and probably generally enhances contrast by interacting with reduced osmium. The binding of osmium by chromatin (Wigglesworth, 1964) probably explains the report by Albersheim and Killias (1963) that bismuth stains DNA in osmium-fixed chromatin. In contrast to section staining, en bloc staining by bismuth is highly specific (Locke and Huie, 1977). En bloc staining with Bi-nitrate-tartrate in a triethanolamine buffer allows bismuth to

THE two main classes of chromatin-associated nuclear proteins appear to be involved with maintaining chromatin structure and regulating gene expression. Histones are small basic proteins soluble in dilute acids. They inhibit transcriptional activity and are thought to be responsible for tightly packing DNA in the nucleus (Lewin, 1974). They form oligomers with each other (an ionic interaction) and interact with DNA both ionically (through their amine and amidine groups) and hydrophobically (Langan and Hohmann, 1975; Lewin, 1974; Russev et al., 1974). Nonhistone proteins (NHPs) are those proteins other than histones that separate with purified DNA (Elgin and Weintraub, 1975). Most are released from DNA in high salt solutions. Some are phosphorylated and concerned with the control of gene expression (Kleinsmith, 1975). The ultrastructural localization of either phosphorylated NHPs or exposed The Cell Science Laboratories, Department of Zoology, University of Western Ontario, London, Canada. * Present address: Dept. of Biology, Carleton University, Ottawa, Ontario, Canada. Received

17 January

1978. 365

366

BROWN

react with two kinds of groups distinguishable by modification of the fixation procedure. Formaldehyde fixation allows Bi binding to some amine and amidine groups and probably to primary phosphates. Glutaraldehyde fixation blocks the basic reactive groups, presumable by cross-linking the amines (Locke and Huie, 1977). In an interphase nucleus, Bi stains some components from euchromatic regions very specifically. Nuclease treatment does not decrease this staining, showing that it is not due to nucleic acids. Nucleolar staining is sensitive to glutaraldehyde crosslinking, while staining of the 50 nm perichromatin and 20 nm interchromatin granules is unaffected by this treatment.

This paper uses isolated mouse liver nuclei to show that bismuth binds to nucleoproteins in situ and that the binding is through primary phosphates and exposed amino groups. Materials and Methods Strain ReJ 129 mice were used as the source of liver tissue for the preparation of intact cells and isolated nuclei. Zsolation of nuclei. Nuclei were isolated using a modification of the procedure of Blobel and Potter (1966). The steps of the isolation, summarized in Table 1, were carried out at 04°C. Excised livers were

Table 1. Isolationof Nuclei Excised Livers TKM-A Slice and rinse Homogenize in 3 volumes of TKM-A

+ Filter through 2 layers of cheesecloth i Add 2 volumes of TKM-C and mix by inversion+homogenate in TKM-B Centrifuge 4~,000 g, 30 min 4°C and discard supernatant

+ Resuspend pellet in 3 volumes of TKM-B and underlay with l/3 volume of TKM-C. Centrifuge 49,000 g, 1 hr, 4°C and discard supernatant. Suspend pellet containing isolated nuclei in 2 volumes of TKM-A.

AND LOCKE

4 Underlay with l/3 volume of TKM-C.

4 Centrifuge at 49,000 g, 30 min. 4°C and discard supematant.

c Resuspend pellet of isolated nuclei in 2 volumes of TKM-A.

NUCLEOPROTEIN

BY BISMUTH

LOCALIZATION

immediately placed in ice-cold buffer containing 0.05 M tris(hydroxymethyl)aminomethane-HCl pH 7.4 (Tris), 5 mM MgCla, 25 mM KCI and 0.25 M sucrose (TKM-A). The tissue was sliced into 1 mm cubes and washed once in the same buffer. Homogenization was carried out with 5-7 strokes of a motor driven teflon pestle at 1700 rpm in 3 volumes of TKM-A. The homogenate was filtered through two layers of cheesecloth and mixed by inversion with two volumes of the same buffer containing 2.3 M sucrose (TKM-C). This mixture, now 1.6 M in sucrose (TKM-B), was centrifuged at 49,000 g for 30 n-tin at 4°C in a Sorval RC-2B refrigerated centrifuge. The supernatant was discarded, and the walls of the centrifuge tubes wiped with a kimwipe. The pellet was resuspended in three volumes of TKM-B and underlaid with l/3 volume of TKM-C. This step gradient was centrifuged at 49,000g for 1 hr at 4°C. Alternatively, the homogenate was mixed with two volumes of TKM-C, underlaid with l/3 volume of TKM-C and centrifuged at 49,000g for 1 hr at 4’C. The supernatant was discarded and the walls of the tubes wiped clean with a kimwipe. The nuclei, suspended in two volumes of TKM-A, wereexamined for cytoplasmic contamination in a Zeiss photomicroscope II at 400x magnification and counted with the aid of a haemocytometer.

367

Further washes for enzyme treatments, alkaline hydrolysis, extraction procedures and preparation of intact nuclei for EM followed centrifugation at 2000g for 10 min (unless otherwise stated) at the designated temperature in not less than four volumes of solution. A Sorval RC-2B refrigerated centrifuge equipped with an SS-34 rotor was used when the temperature was 4°C. A Sorval GLC-1 centrifuge equipped with an HL-4 swinging bucket rotor (50 ml buckets) was used for centrifugation at room temperature. All enzyme digestion experiments, alkaline hydrolysis and salt and acid extraction together with samples incubated in buffer were repeated at least three times. The effects on Bi staining were consistent for each treatment. Salt

and acid extractions (Procedure summarized in Table 2). Isolated nuclei, suspended in TKM-A and divided into six equal fractions by volume, were washed once in TKM-A containing 10-a M phenylmethanesulfonyl fluoride, to inhibit protease activity, and then washed three times in six volumes of the desired extraction solution at 4°C for 2.5-3.5 hr with constant shaking. The following extraction solutions were used :

(I) 0.14 M salt: 0.14 M NaCl in 0.01 M Tris-HCI pH 7.2 at room temperature

Table 2. Salt and acid extractions of isolated nuclei ~~~

Extraction solutions

~.

Fix in Caco-buffered Wash in ~~~~. _~~~~ 4 % formaldehyde 5 % glutaraldehyde CKM

0.14 M NaCl

X

0.34 M NaCl

X

Figure

X

X

28

X

X

X

29

X

X

30

X

X

31

X

X

32

x

X X

0.14

Osmium staining

X

X

0.6 M NaCl

Bismuth staining

M NaCl

X

-

X

X

33

_

+

x

x

34

X

-

X

X

35

X

X

36

x 0.25

N HCI

0.6 M NaCl 0.14 M NaCl 0.25 N HCl 24

X x

BROWN

368

LOCKE

in 0.01 M sodium phosphate buffer pH 7.1 containing 0.1 ‘A SDS, 0.1 oA P-mercaptoethanol and 0.25 M sucrose were loaded on top of the gels. The stacking gel was 3% in acrylamide in 1 M Tris-HCl buffer pH 6.8 containing 0.8 % SDS. The separating gel was 8.7% in acrylamide in 3 M TrisHCl pH 8.9 containing 0.8 % SDS. Separation was achieved at a constant current of 1.5 mA/tube. Densitometer tracings were done in an Isco gel scanner, absorbance monitor and model 612 chart recorder at a chart speed of 1 in./min and absorbance setting 1.0.

(II) 0.34 M salt: 0.34 M NaCl in 0.01 M Tris-HCl pH 7.2 at room temperature (III) 0.6 M salt: 0.6 M NaCl in 0.01 M Tris-HCl pH 7.2 at room temperature (IV) Low salt and acid: 0.14 M NaCl in 0.25 N HCI, pH 1.0 at room temperature The nuclei were pelleted by centrifugation at 6000 g for 20 min at 4°C and the supernatants saved for protein analysis by SDSpolyacrylamide gel electrophoresis. The nuclear pellet was suspended in six volumes of 0.05 M NaCaco-HCl buffer pH 7.4 containing 5 mM MgCla and 25 mM KC1 (CKM) and prepared for EM.

Enzyme

treatments

and alkaline

hydrolysis

(summarized in Table 3). Following isolation, nuclei were washed three times in CKM at 4°C. They were then suspended in 4% formaldehyde in 0.1 M NaCaco-HCl pH 7.4 containing 4% sucrose and fixed for 30 min at 4°C. Three washes in 0.1 M NaCaco-HCl pH 7.4 containing 4% sucrose (CS) followed,

This protocol is a modification of the procedure of Laemmli (1970). Protein concentration was determined by a modified Hartree (1972) procedure. 100 and 200 pg of protein from the salt extractions and 50 and 100 pg of protein from the acid extractions solubilized

SDS-Polyacrylamide

AND

gel electrophoresis.

Table 3. Enzyme treatments and alkaline hydrolysis of isolated nuclei Isolated nuclei 4 Wash in CKM J Brief fix in Caco-buffered 4 Proteinase K digestion ~_____

4 % formaldehyde

i Alkaline hydrolysis i

J

4

FIX IN CACO-BUFFERED I

1 I_

formaldehyde

4

7 ,

4 Alkaline phosphatase digestion

formaldehyde glutaraldehyde

4

~~ _~ ~~~~

:

J

formaldehyde glutaraldehyde

_J_~

4

Incubate in Bi solution 4 ___~J.. ..~ Postfix and stain with 0~04

F1 J

~.

4

~

$ Dehydrate,

4 embed and section

4

J FIGURE

6-9 (+UA) I_ _I__

17

4

~

i

4

4

21

23

I i

-1 ~~ __L__~_ J-----~

:

18

~

NUCLEOPROTEIN

LOCALIZATION

BY BISMUTH

after which the nuclei were washed in the enzyme buffer. The procedures for the individual enzyme treatments and alkaline hydrolysis are as follows: (1) Proteinuse K obtained from E. Merck, Darmstadt, Germany. Wash three times in TKM-A at room temperature. Incubate lo6 nuclei/ml in 50 pg/ml proteinase K in the same buffer at 37°C for I-2.5 hr with mild agitation. Wash three times in TKM-A adjusted to pH 8.8 at 4°C. (This hydrogen ion concentration places the enzyme at its isoelectric point (Merck) thus ensuring its removal from the nuclei and preventing the possibility of artifactual staining due to its continued presence.) (2) Alkaline phosphatase obtained from Sigma, type III-R, E. coli substantially RNase and DNase free. Wash three times in 0.25 M Tris-HCl pH 8.0 at room temperature. Freeze-thaw the nuclei three times to facilitate enzyme penetration. Incubate IO6 nuclei/ml in 5 pg/ml alkaline phosphatase in the same buffer at 37°C for 1 hr with mild agitation. Test the supernatant for the presence of inorganic phosphates by the method of Pollard and Korn (1973). Wash the nuclei three times in the same buffer at 4°C. The activity of this enzyme was always tested immediately prior to incubation with the nuclei by adding 0.1 ml of the enzyme solution (5 pg/ml protein in distilled water) to 3 ml of 0.001 M p-nitrophenyl phosphate in 1.0 M Tris-HCl pH 8.0. Changes in the absorbance were measured at 410 nm. The specific activity, calculated by the following formula, was always above 20 units/mg protein under the conditions described. Units of Activity_ mg of protein change in AJto/min x 1000 1.62 x 1O-’ x mg enzyme/ml reactton mixture (Worthington.

1972).

(3) Alkaline hydrolysis. Fixed nuclei were washed three times in 35 min in 1 N KOH

369

pH 13.5 and incubated in the KOH solution for 1 hr at 37°C. After the treatments or the washes following the treatments, the nuclei were suspended in CS and prepared for EM as outlined below. Electron microscopy. Mice were killed by cervical dislocation and excised livers were immediately placed in an ice-cold saline solution (0.14 M salt). The tissue was sliced into 1 mm cubes, rinsed once in saline and then fixed overnight in 5% formaldehyde in 0.05 M NaCaco buffer adjusted to pH 7.4 with HCI, containing 4% sucrose. Isolated nuclei were washed in either CKM or CS and fixed at 4°C for l-3 hr in: (1) 4 % formaldehyde in CS, (2) 5% glutaraldehyde in 0.1 M NaCaco-HCl, pH 7.4 containing 2y, sucrose or, (3) 4% formaldehyde in TKM pH 7.4 containing 4% sucrose. Isolated nuclei or tissue blocks were washed four times in buffer, stained with Bi at pH 7.0 (Locke and Huie, 1977), postflxed in 1 “/;; osmium in 0.05 M NaCaco-HCl pH 7.4 containing 4% sucrose, washed in three changes of distilled HaO, and stained in 2.5 ‘A aqueous uranyl acetate (UA) for 2 hr at 60°C (Locke et al., 1971). The tissue or pelleted nuclei were then washed three times in distilled water, dehydrated in a graded ethanol series, washed in propylene oxide, and embedded in Araldite epoxy resin. Sections were cut on either glass or diamond knives with an OMU-3 ultramicrotome to a standard thickness giving silver to gold interference colours (approximately 850 A). Consistency of density and contrast in electron microscopy and photography. Since most of this work involves direct comparison of Bi staining intensity between control and experimental conditions, features affecting density and contrast were made as consistent as possible within a given experiment and, when possible, from experiment to experiment. All EM work was done on a Philips 300 electron microscope at 80 kV with an objective aperture of constant size (20 pm). One plate magnification of 25,000 was used for comparison. The EM plates were either Kodak#4489 or Kodak#SO 163, but only one type was used within each expertment. For preparations stained with osmium, bis-

370

BROWN

muth or bismuth-osmium, a constant electron exposure was used and plates and prints were given the same treatment. Results Morphology

of liver nuclei in intact cells.

Uranyl acetate en Hoc staining (Fig. 1) shows mouse liver nuclei with well developed nucleoli and heterochromatin particularly in masses near the nuclear membrane. Euchromatin contains interchromatin granules and occupies much of the nucleoplasmic space out to the nuclear pores. Preparations stained with Bi and osmium (Fig. 2) demonstrate specific binding of Bi to the nucleolus and to interchromatin granules (ICGs). Isolated nuclei.

nuclear

UA staining morphology is little

shows that changed by

LOCKE

isolation (Fig. 3), although interchromatinic spaces are less dense allowing clear distinction between euchromatin, heterochromatin and the nucleolus. Bi-osmium staining is also unaffected by the isolation procedures (Figs. 4, 5). Fig. 4 shows the specific Bi staining of the nucleolus and the ICGs. Fig. 5 shows a number of isolated nuclei which remain for the most part intact. There is some membrane contamination and the few ribosomes are associated with the outer nuclear membrane. Osmium contributes little to the density of Figs. 4, 5. When isolated nuclei are stained only with osmium, the nuclear membranes and the nucleoplasm have a faint grey background just sufficient to allow distinction between dispersed and condensed chromatin, but not between condensed chromatin and the nucleolus. Bismuth

Abbreviations bovine serum albumin BSA sodium cacodylate Caco condensed chromatin cc CKM 0.05 M NaCaco-HC1 buffer pH 7.4 containing 25 mM KC1 dc dispersed chromatin euchromatin EU He heterochromatin ICGs interchromatin granules nuclear pore np nucleolus NU tris(hydroxymethyl)aminomethane Tris uranyl acetate UA Electronmicrographs-All

AND

5 mM MgCla and

except 1, 2 and 5 are magnified

x 54,500.

Fig. 1. The morphology of mouse liver cell nuclei stained with uranyl acetate. Nucleoli are well developed, heterochromatin is located in masses around the nuclear periphery and euchromatin, occupying much of the nucleoplasmic space, contains 20 nm interchromatin granules. Treatment: Caco-formaldehyde, 0~04, uranyl acetate. x 23,000. Fig. 2. The morphology of mouse liver cell nuclei stained with bismuth. The staining is specific for the nucleolus and interchromatin granules. Treatment: Caco-formaldehyde, bismuth, OsOa. x 23,000. Figs. 3, 4. Isolated

mouse liver nuclei maintain

their normal

morphology.

Fig. 3. The nucleolus, heterochromatin and ICGs are easily resolved with uranyl acetate staining. Treatment: Caco-formaldehyde, 0~01, uranyl acetate, UA and lead. X 54,500. Fig. 4. The nucleolus and ICGs are specifically Caco-formaldehyde, bismuth, 0~04. x 54,500.

stained

with bismuth.

Treatment:

372

BROWN

AND

LOCKE

Fig. 5. Isolated nuclear fractions contain very little contamination. Nuclei have remained intact and there are some membrane fragments visible. Treatment: Cacoformaldehyde, bismuth, 0~04. x 17,000. staining on its own easily distinguishes between the closely packed, finely granular staining of the nucleolus and the dispersed, dense staining of the ICGs in euchromatinic areas. Combined Bi-osmium staining thus allows convenient and reliable distinction between euchromatic, heterochromatic and nucleolar regions where osmium does not interfere with the bismuth localizations. The specificity of bismuth staining. Since bismuth staining in the nucleus is insensitive to nuclease treatments (Locke and Huie, 1977), the interaction is presumed to involve nucleoproteins. Isolated, fixed nuclei were

digested with Proteinase K to demonstrate Bi affinity for nucleoproteins in situ. Figs. 6, 7 show the difference between an osmiumUA stained nucleus after incubation in buffer for 2.5 hr at 37°C (Fig. 6) and one incubated with Proteinase K under the same conditions (Fig. 7). Euchromatic areas and the nucleolus were removed sequentially by the digestion which changed the condensed chromatin to a matrix of 5-10 nm fibers. The enzyme causes complete removal of material having an affinity for Bi (Figs. 8, 9), confirming the idea that Bi binds to nucleoproteins in situ using the staining conditions employed.

NUCLEOPROTEIN

LOCALIZATION

BY

Bismuth binding to amino groups

Bi staining is through an interaction with amino groups is confirmed in the following way. Tissue was fixed in Tris-buffered-

Amino group crosslinking. Some Bi staining is sensitive to glutaraldehyde crosslinking (Fig. 16), indicating the involvement of amino groups in Bi staining (Locke and Huie, 1977). The crosslinking reaction, in its simplest form, is 2R-NHa

formaldehyde (thereby providing an external supply of free cc-amino groups) to see if the limitation of a-amino group contiguity was the basis for increased Bi binding in formaldehyde, as opposed to glutaraldehyde fixed tissue. Figs. lo-13 compare Bi staining of isolated nuclei fixed in cacodylate-buffered formaldehyde (Fig. lo), cacodylate-buffered glutaraldehyde (Fig. 11) and Tris-buffered formaldehyde (Fig. 12). The presence of free cc-amino groups in conjunction with formaldehyde fixation blocks some staining of the nucleolus and ICGs. Section staining with UA and lead (Fig. 13), shows that the loss of Bi nucleolar and ICG staining is not due to extraction.

+ HC-CHzCHzCHa---CH+

I!

0 R-NH-CHCH2CHaCHzCH==N-R f Ha0

373

BISMUTH

+ OH-

with either single or double aldehyde-amine bonds being formed (Hayat, 1970). Formaldehyde also interacts with amino groups, but most of the reactions are reversible (Hayat, 1970). They are summarized as follows:

(I) RH + CHzO ++ RCHzOH + RH H R-CHe-R. Alkaline hydrolysis. Incubation

(2) -NH2 + CH20 ++ N=CHz + Hz0 + CHzO + -N-CHCHzOH c RH H -N =CHCH2-R where R is -NHa, =NH or -CONH.

of unfixed

nuclei in a solution of high pH should extract much of the RNA (Murphy and Bonner, 1975), at least 70 % of the NHPs and 82-84 % of the histones (Murphy and Bonner, 1975; Russev et al., 1974). The procedure should unmask all of the primary amino groups on histone HI, 55% of the primary amino groups on histones H2A and H2B and 25:/ of the primary amino groups on histones H3 and H4 (Malchy and Kaplan, 1976). Since the preparations used here were

Irreversible crosslinking is dependant upon the close apposition of a-amino groups. Glutaraldehyde, however, can form polymers with itself and thereby acheive crosslinking without the limitation of a-amino group contiguity. The idea that some of the Table 4. Amino group cross/inking in isolated nuclei

Isolated nuclei in TKM i Wash in CKM

,

+ Fix in Tris-buffered formaldehyde 4~

~_~~

4,

Fix in Caco-buffered formaldehyde

~~~__

4,

Fix in Caco-buffered glutaraldehyde m-k-

_A_ ~~_. ~~_~ Incubate in Bi solution

1

_~

~ )

L__

$__ -Postfix

-1-L

_~~~

_~

A

and stain with osmium

J

-

.~~..

~__ ._ -4

!

Dehydrate, embed and section

I ~~_. y-

_~~~

_A

._~

I..

~~~_

_L__-_..

FIGURES

14, lS(+UA)

4, s, 12

13

I

374

BROWN

fixed prior to alkaline hydrolysis, CIOSSlinking of the nucleoproteins should prevent appreciable extraction while allowing alkaline hydrolysis to expose primary amino groups. The result is an increased Bi-ICG staining (Figs. 14, 15) that can be blocked by glutaraldehyde (Fig. 17), confirming the hypothesis that some Bi is binding to amino groups exposed by alkaline hydrolysis.

AND

LOCKE

Bismuth staining of primary phosphate groups. Bismuth stains primary phosphate groups in spot tests (Locke and Huie, 1977). Isolated, fixed nuclei were therefore incubated in a solution containing alkaline phosphatase in an attempt to remove the glutaraldehyde insensitive localizations seen in the nucleus. After the incubation with enzyme, the supernatants were tested for the release of

Figs. 6-9. Proteinase K removes bismuth staining material. The enzyme sequentially extracts euchromatin and the nucleolus with the subsequent dispersal of condensed chromatin. (Table 3). Fig. 6. Uranyl acetate stained nucleus following incubation in buffer for 2.5 hr at 37°C. Treatment: Caco-formaldehyde. TKM-A, 0~04, UA. x 54,500. Fig. 7. Uranyl acetate stained nucleus showing extraction of euchromatin and the nucleolus and dispersal of condensed chromatin following incubation with Proteinase K for 2.5 hr at 37°C. Treatment: Caco-formaldehyde, Proteinase K, 0~04, UA. x 54,500. Fig. 8. Bismuth stained nucleus following incubation in buffer for 2.5 hr at 37°C. Nucleoli and ICGs stain specifically. Treatment: Caco-formaldehyde, TKM-A, bismuth, 0~04. x 54,500. Fig. 9. All material having an affinity for bismuth is removed by incubation with Proteinase K for 2.5 hr at 37°C. Treatment: Proteinase K, bismuth, 0~04. x 54,500. Figs. 10-13. Amino

group crosslinking

Fig. 10. Specific staining of nucleoli formaldehyde, bismuth, 0~04. x 54,500. Fig. 11. Glutaraldehyde is unaffected. Treatment:

decreases and

crosslinking blocks Caco-glutaraldehyde,

ICGs

staining

from isolated nuclei Caco-formaldehyde,

by bismuth.

by bismuth.

(Table 4)

Treatment:

Caco-

Bi-nucleolar staining. Bi-ICG bismuth, 0~04. x 54,500.

staining

Fig. 12. Fixation with Tris-buffered formaldehyde blocks Bi-nucleolar staining and decreases Bi-ICG staining. Treatment: Tris-formaldehyde, bismuth, 0~04. x 54,500. Fig. 13. Uranyl acetate and lead section staining shows that fixation in Tris-buffered formaldehyde has not allowed extraction of nuclear components. Treatment: Trisformaldehyde, bismuth, 0~04, UA and lead. x 54,500. Figs. 14-17. Alkaline hydrolysis of fixed nuclei intensifies Bi-ICG sumably by increasing the availability of a-amino groups. (Table 3) Fig. 14. Specific staining ment: Caco-formaldehyde,

staining

pre-

of nucleoli and ICGs in isolated nuclei by bismuth. TreatCKM, Caco-formaldehyde, bismuth, 0~04. x 54,500.

Fig. 1.5. Alkaline hydrolysis increases Bi-ICG staining. Treatment: hyde, IN-KOH, Caco-formaldehyde, bismuth, 0~04. x 54,500.

Caco-formalde-

Fig. 16. Glutaraldehyde blocks hyde, CKM, Caco-glutaraldehyde,

Caco-formalde-

Bi-nucleolar staining. Treatment: bismuth, 0~04. x 54,500.

Fig. 17. Glutaraldehyde blocks the increase in Bi-ICG staining resulting from alkaline hydrolysis. Treatment: Caco-formaldehyde, lN-KOH, Caco-glutaraldehyde, bismuth, 0~04. x 54,500.

010

378

BROWN

AND

LOCKE

Table 5. Quantitative analysis of proteins solubilized by salt and acid solutions Extraction procedure

Amount of protein extracted

0.14 M NaCl

0.34 M NaCl

0.6 M NaCl

0.23 mg

0.66 mg

0.67 mg

1.02mg

21%

60%

61%

92%

% of extractable protein removed*

0.14 M NaCl 0.25 N HCl

0.6 M NaCl 0.14 M NaCl 0.25 N HCI 0.67 +0.43 l.lOmg 61.1 +3s.9 100%

* That is, those which are extractable by the procedures employed. inorganic phosphates. Analysis demonstrated that significant amounts of inorganic phosphates were released by the enzyme treatment. Figs. 18, 19 show that the phosphatase seems to have removed little Bi stainable material from ICGs. However, glutaraldehyde fixed, Bi-osmium stained nuclei show negligible Bi staining of ICGs following exposure to enzyme (Figs. 20, 21). Thus, although incubation in a solution containing alkaline phosphatase does not remove ICGs it does eliminate the glutaraldehyde insensitive Bi-staining component of these granules, as we should expect if Bi binds to primary phosphate groups as well as amino groups on ICGs. Bismuth staining of nucleoprotein

Figs. staining

fractions.

In

an effort to determine which classes of nucleoproteins are stained by bismuth, isolated nuclei were extracted by procedures which selectively removed specific groups of nucleoproteins. The nucleoproteins extracted were analyzed by SDS-polyacrylamide gel electrophoresis and the effect of the extractions on Bi staining of nuclei was observed by electron microscopy Quantitative analysis of extracted nucleoproteins. Table 5 compares the amount of

proteins extracted by various procedures in two pooled experiments. 0.14 M NaCl removed only a small proportion (21%) of the proteins which were extracted by the other procedures and there was little difference between extraction with 0.34 M and 0.6 M

18-21. Alkaline phosphatase removes component of ICGs. (Table 3)

the

glutaraldehyde-insensitive

Bi-

Fig. 18. Incubation in buffer does not change the Bi staining characteristics. Treatment: Caco-formaldehyde, Tris, Caco-formaldehyde, bismuth, 0~04. x 54,500. Fig. 19. Alkaline phosphatase does not change the Bi staining characteristics in formaldehyde fixed nuclei. Treatment: Caco-formaldehyde, alkaline phosphatase, Caco-formaldehyde, bismuth, 0~04. x 54,500. Fig. 20. Incubation in buffer does not change the Bi-staining characteristics of glutaraldehyde-fixed nuclei. Treatment: Caco-formaldehyde, Tris, Caco-glutaraldehyde, bismuth, 0~04. x 54,500. Fig. 21. Glutaraldehyde blocks both nucleolar and ICG staining after incubation with alkaline phosphatase. Treatment: Caco-formaldehyde, alkaline phosphatase, Caco-glutaraldehyde, bismuth, 0~04. x 54,500.

0

18

BROWN 5

AND

LOCKE

6

actin fTOpOmyorin

Fig. 22. The qualitative analysis of proteins extracted from isolated nuclei in salt and acid solutions. Proteins extracted in 0.14 M NaCl (gel 1), 0.34 M NaCl (gel 2), 0.6 M NaCl (gel 3) 0.14 M NaCl in O-25 N HCl (gel 4) and 0.14 M NaCl in 0.25 N HCl after extraction with 0.6 M NaCl (gel 5) were solubilized in SDS and seoarated on the basis of molecular weight through SDS-polyacrylamide gels. Gel 6 contained myosin (molecular weight 200,000), actin (molecular weight 42,000), and RNase (molecular weight 13,500) which acted as standards for estimation of the molecular weights of protein bands on the sample gels.

NaCl (60 and 61% respectively). Low salt and acid removed most of the extractable proteins (92 %) . QuaIitative analysis: gel electrophoresis.

S DS-polyacrylamide

Figs. 22, 23 show few qualitative differences between the species of proteins extracted with the various salt solutions. 0.14 M and 0.34 M salt solubilized the same proteins since the number of bands on the gels (gels 1 and 2; Fig. 22) and peaks on the scans (the top two scans in Fig. 23) remained constant. However, extraction with 0.6 M salt solubilized, to a greater degree, two distinct high molecular weight protein species (bands 1 and 2, gel 3 or, peaks 1 and 2; Figs. 22, 23) and more of the proteins comprising band and peak 18. When considering this data, in conjunction with the

quantitative results, one would surmise that 0.34 M salt solubilizes the same proteins as 0.14 M salt (the same number of bands appear on the gels) but does so to a greater extent (60% of the extractable proteins are removed by 0.34 M salt as compared to 21% by 0.14 M salt). 0.6 M salt, in addition to solubilizing the same proteins as the other two salt solutions and to approximately the same extent as 0.34 M salt, extracted two high molecular weight protein species (bands 1 and 2) as well as proteins having a molecular weight of about 46,500 (band 18) to the degree that they become distinct bands with this method of analysis. Extraction with salt and acid removes mainly histones (bands 21 to 25, gel 4, Fig. 22) with trace amounts of NHPs (bands l-20). Most of the NHPs are removed by prior extraction with 0.6 M salt as were some of

NUCLEOPROTEIN

LOCALIZATION

BY BISMUTH

the histones (gel 5, Fig. 22). The salt and acid extracts do not contain either the high molecular weight proteins (band 1 and 2) or proteins constituting band 18. Direct comparison of the gels containing salt extracted proteins is possible because the gels 1,2 and 3 were each loaded with 200 pg of protein. Gels 4 and 5 each contain 100 pg of protein and so these also can be directly compared. \

Variations

in Species

of Salt

XJ

w

NHPr

70

MOLECULAR (daltons

80

The results show that: 1. 0.34 M salt extracted the same proteins as 0.14 M salt but to a greater extent, 2. 0.6 M salt extracted proteins comprising three additional distinct bands (bands 1, 2 and 18), and 3. salt and acid extraction removed mainly histones.

Solubilized

and Acid

loo

381

by Different

Salt

and

Solutions

17.0

160

200

WEIGHT x l@)

Fig. 23. Densitometer scans of the acidic proteins extracted from the isolated nuclei by salt and acid solutions. The scans are of gels l-5 (Fig. 22). The numbers on the peaks correspond to the numbers on the gels. The scale of the molecular weights is derived from the position of the protein standards appearing on gel 6 (Fig. 22).

BROWN AND LOCKE

382

Knowing that some of the extractions could be related to removal of particular proteins, it became important to observe stained nuclei, to see if the removal could be correlated with any particular reduction of bismuth staining. Microscopical observations. Extraction with 0.14 M salt caused no alteration in Bi staining (Fig. 24 vs 26). Incubation with

0.34 M salt decreased the nucleolar staining, probably by the extraction of ribonucleoprotein particles, but had no effect on Bi-ICG staining in either formaldehyde or glutaraldehyde fixed nuclei (Figs. 27, 28). After 0.6 M salt extraction, the nucleolus contained few Bi-staining components (Fig. 29). The ICG staining was decreased in formaldehyde fixed nuclei (Fig. 24 vs 29) and absent in those fixed in glutaraldehyde (Fig. 30). Since 0.6 M

Figs. 24-26. 0.14 M salt extraction nuclei. (Table 2 and 6)

does not affect bismuth

Fig. 24. Incubation in buffer does not affect bismuth Caco-formaldehyde, bismuth, 0~04. x 54,500.

staining

staining.

in isolated

Treatment:

Fig. 25. Incubation in buffer does not affect the glutaraldehyde-insensitive of ICC&. Treatment: Tris, Caco-glutaraldehyde, bismuth, 0~04. x 54,500.

Tris, staining

Fig. 26. 0.14 M salt extraction does not affect Bi-nucleolar Treatment: 0.14 M NaCl in Tris, Caco-formaldehyde, bismuth,

or Bi-ICG staining. 0~04. x 54,500.

Figs. 27 and 28. 0.34 M salt extraction affect Bi-ICG staining. (Table 2 and 6).

staining

Fig. 27. Treatment: x 54,500.

0.34 M NaCl

Fig. 28. Treatment: x 54,500.

0.34 M NaCl

decreases

in Tris,

nucleolar

but does not

Caco-formaldehyde,

bismuth,

0~04.

in Tris, Caco-glutaraldehyde,

bismuth,

0~04.

Figs. 29 and 30. 0.6 M salt extraction insensitive ICC staining. (Table 2 and 6)

reduces

nucleolar

and

glutaraldehyde-

Fig. 29. Treatment: x 54,500.

0.6 M NaCl

in Tris,

Caco-formaldehyde,

bismuth,

0~04.

Fig. 30. Treatment: X 54,500.

0.6 M NaCl

in Tris,

Caco-glutaraldehyde,

bismuth,

0~04.

Figs. 31 and 32. Extraction with salt and acid disperses chromatin fibers, removes the Bi-staining component of the nucleolus and reduces the intensity of Bi-ICG staining in isolated nuclei. (Table 2 and 6) Fig. 31. Treatment: x 54,500.

0.14 M NaCl

in 0.25 N HCI, Caco-formaldehyde,

bismuth,

Fig. 32. Treatment: x 54,500.

0.14 M NaCl

in 0.25 N HCI, Caco-glutaraldehyde,

bismuth,

Fig. 32. Treatment: x 54,500.

0.14 M NaCl

in 0.25 N HCI, Caco-glutaraldehyde,

bismuth,

oso4.

oso4.

oso4.

Figs. 33 and 34. Extraction with high salt followed by salt and acid removes Bi-staining components.from isolated nuclei. (Table 2 and 6) Fig. 33. Treatment: 0.6 M NaCl, 0.14 M NaCl in 0.25 N HCI, Caco-formaldehyde, bismuth, 0~04. x 54,500. Fig. 34. Treatment: 0.6 M NaCI, 0.14 M NaCl in 0.25 N HCI, Caco-glutaraldehyde, bismuth, 0~04. x 54,500.

all

c 1.

,

*

-

BROWN

386

salt selectively extracts proteins comprising bands 1, 2 and 18, we may presume that they are responsible for the glutaraldehydeinsensitive staining. The persistence of BiICC staining in formaldehyde-fixed nuclei after 0.6 M salt extraction indicates that either (1) this treatment exposes primary amino groups not previously available for interaction with Bi (since the staining is glutaraldehyde-sensitive) or, (2) some of the Bi-ICG staining normally involves primary amino groups. The latter interpretation is supported by the alkaline phosphatase experiments where ICG staining persisted in formaldehyde-fixed nuclei after enzyme treatment but was blocked by glutaraldehyde. Up to this point in the extraction procedure, the nuclei maintained a fairly normal morphology. Salt and acid transformed the densely packed heterochromatin into dispersed fibers (Figs. 31, 32) and reduced the ICG staining in both formaldehydefixed and glutaraldehyde-fixed nuclei (Figs 31, 32 vs Figs. 24, 25). Thus the salt and acid extraction that removes histones, also removes the glutaraldehyde-sensitive Bi-staining component of ICGs. If nuclei are incubated in salt and acid after extraction with 0.6 M salt, all Bi staining is lost (Figs. 33, 34). This confirms the idea that 0.6 M salt solubilizes proteins that are responsible for the glutaraldehyde insensitive Bi-ICG localization, and confirms the suggestion that salt and acid extracts the proteins responsible for glutaraldehydesensitive Bi-ICG staining. The effects of the different extraction procedures are summarized in Table 6. Conclusions and Discussion The results allow the following Table 6. Summary

Treatment Fixative

conclusions:

Form.

Bi staining of the nucleolus

+++

Bi staining of ICGs

+++

Glut. ++++++

0.34 M Salt Form. ++

Glut. ++

LOCKE

1. Bismuth stains nucleoproteins in euchromatic regions of isolated interphase mouse liver nuclei since staining is sensitive to Proteinase K digestion. 2. Glutaraldehyde-sensitive staining involves a Bi interaction with amino groups since it is reduced (Bi-ICG staining) or almost eliminated (Bi-nucleolar staining) by amino group crosslinking and increased by exposing amino groups. 3. Glutaraldehyde-insensitive staining of ICGs involves an interaction with primary phosphate groups since all staining is eliminated by alkaline phosphatase digestion and glutaraldehyde fixation. 4. The Bi-ICG interaction involves both primary phosphate groups and amino groups since staining persists in isolated nuclei after incubation with alkaline phosphatase and formaldehyde fixation but staining is blocked when similar nuclei are fixed in glutaraldehyde. 5. The glutaraldehyde-insensitive Bi-ICG staining probably involves a Bi interaction with phosphorylated NHPs since NHPs are the only new species of nucleoprotein solubilized when the glutaraldehyde-insensitive, alkaline phosphatase-sensitive staining is removed by salt extraction. 6. The glutaraldehyde-sensitive Bi-ICG staining probably involves a Bi interaction with histones since this localization is

eliminated only after an acid extraction that mainly removes histones (gel 4, Fig. 22; Brasch et al., 1972). This study was undertaken in an attempt to localize nucleoproteins ultrastructurally at sites of transcriptional activity. Bismuth binding to either exposed amino groups or primary phosphate groups, currently believed to be concentrated at these sites of activity (Langan and Hohmann, 1975; Lewin, 1974;

of the effects of the various extraction

0.14 M Salt

AND

procedures

0.6 M Salt Form. +/_ ++

Glut.

on bi-staining

Salt and acid Form.

Glut.

of isolated

nuclei

Salt and acid after 0.6 M salt Form.

Glut.

-

-

-

-

_

-

+

+

-

-

NUCLEOPROTEIN

LOCALIZATION

BY

Russev et al., 1974; Kleinsmith, 1975), is proposed as the basis for this localization. Results from the crosslinking and alkaline hydrolysis experiments show that bismuth interacts with exposed amino groups. The sensitivity of the Bi-ICG staining to both alkaline phosphatase and glutaraldehyde crosslinking indicates that this localization involves interactions with both amino groups and primary phosphate groups. The extraction experiments show that: (1) the glutaraldehyde-sensitive Bi-ICG interaction involves nucleoproteins extractable in salt and acid, (2) the glutaraldehyde-insensitive BiICG staining probably results from an interaction with phosphorylated NHPs, and (3) the nucleolar staining is probably the result of a Bi interaction with ribonucleoprotein particles. The results may relate the ultrastructural localization of these proteins to what is known of their function. Histones decrease the transcriptional activity of chromatin (Lewin, 1974; Kitzis, 1976) and their interaction with DNA involves amino groups (Langan and Hohmann, 1975; Lewin, 1974; Russev et al., 1974). Glutaraldehydesensitive Bi localizations on ICGs may

387

BISMUTH

therefore pinpoint the position of histones which are not tightly bound to DNA through these groups and which may not be inhibiting transcriptional activity at these sites. Although extraction with salt and acid mainly removes histones (Fig. 22), some acid-soluble NHPs are present in the gels and Bi may be interacting with reactive amino groups on these NHPs at the ICGs. Phosphorylation of NHPs is correlated with increased transcriptional activity (Kleinsmith, 1975) and so the glutaraldehydeinsensitive Bi-ICG staining may localize phosphorylated NHPs mediating this activity. Bismuth staining, because of its specificity, realizes the resolving capability inherent in the principle of heavy metal staining. If the argument presented is correct, the procedure is capable of resolving individual transscriptionally active sites. Acknowledgements Strain ReJ 129 mice were kindly donated by Dr B. G. Atkinson under a grant from the Muscular Dystrophy Foundation of Canada. This work was supported by N.R.C. Grant #A6607 to M.L.

References AINSWORTH, S. K., ITO, S. and KARNOVSKY, M. J. 1972. Alkaline Bismuth Reagent for High Resolution Ultrastructural Demonstration of Periodate-reactive Sites. J. Histochem. Cytochem., 20 (12), 995-1005. AINSWORTH, S. K. and KARNOVSKY, M. J. 1972. An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections. J. Histochem. Cytochem., 20 (3), 225-229. ALBERSHEIM,P. and KILLIAS, U. 1963. The use of bismuth as an electron stain for nucleic acids. J. Cell Biol., 17 (l), 93-103. BARRET, J. M., HEIDGER,P. M. and KENNEDY, S. W. 1975. Chelated bismuth as a stain in electron microscopy. J. Histochrm. Cytochem., 23 (lo), 780-787. BLOBEL, G. and POTTER, V. R. 1966. Nuclei from rat liver: isolation method that combines purity with high yield. Science, 154, 1662-1665. BRASCH, K., SETTERFIELD,G. and NEELIN, J. M. 1972. Effects of sequential extraction of histone proteins on structural organization of avian erythrocyte and liver nuclei. Exp. Ccl/ Rrs., 74, 27-41. ELGTN, S. and WEIN~RAUB, H. 1975. Chromosomal proteins and chromatin structure. A. Rev. Biochrm.,

44,725-775. HARTREE, E. F. 1972. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt. Biochem., 48, 422427. HAYAT, M. A. 1970. Principles and Techniques of Electron Microscopy. Biological Applications. Vol. 1, pp. 13-95, 241-302. Van Nostrand Reinhold Co., New York. KITZIS, A., DEFER, N., DASTUGUE, B., SABATIER, M. and KRUH, I. 1976. Effect of heparin on chromatin.

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KLEINSMITH,L. J. 1975. Do phosphorylated proteins regulate gene activity? In Chromosomal Proteins and Their Role in the Regulation of Gene Expression. (eds. G. S. Stein and L. J. Kleinsmith), pp. 45-57. Academic Press Inc., New York. LAEMMLI,U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage Tq. Nature, Lond., 227, 680-685. LANGAN, T. and HOHMANN, P. 1975. Analysis of specific phosphorylation sites in lysine rich (Hl) histone: An approach to the determination of structural chromosomal protein functions. In Chromosomal Proteins and Their Role in the Regulation of Gene Expression. (eds. G. S. Stein and L. J. Kleinsmith), pp. 113-125. Academic Press Inc., New York. LEWIN, B. 1974. Gene Expression. Vol. 2 Eucaryotic Chromosomes, pp. 101-147. John Wiley and Sons Ltd., Toronto, Canada. LOCKE, M. and HUIE, P. 1975. The Golgi complex/endoplasmic reticulum transition region has rings of beads. Science, 188, 1219-1221. LOCKE, M. and HUIE, P. 1976a. The beads in the Golgi complex/endoplasmic reticulum region. J. Cell Biol., 70, 384-394. LOCKE, M. and HUIE, P. 1976b. Nucleoprotein localizations by bismuth staining. Proc. Mirrosc. Sot. Can., III 96-97. LOCKE, M. and HUIE, P. 1977. Bismuth staining for light and electron microscopy. Tissue & Cell, 9 (2) 347-371. LOCKE, M., KRISHNAN, N. and MCMAHON, J. T. 1971. A routine method for obtaining high contrast without staining sections. .7. Ceil Biol., 50, 540-544. MALCHY, B. L. and KAPLAN, H. 1976. Unmasking of histone amino groups in chromatin at high pH. Biochem J., 159, 173-175. MERCK, E. Proteinase K. A New Protease with Remarkable Properties. E.M. Reagents, BDH Chemicals, Toronto, Canada. MURPHY, R. F. and BONNER, J. 1975. Alkaline extraction of non-histone proteins from rat liver chromatin. Biochim. biophys. Acta, 405, 62-66. POLLARD, T. D. and KORN, E. D. 1973. Acanthamoeba Myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J. biol. Chem., 248, 46824690. RIVA, A. 1974. A simple and rapid staining method for enhancing the contrast of tissues previously treated with uranyl acetate. J. Microsc., 19, 105-107. RLJSSEV,G., VENKOV, C. and TSANEV, R. 1974. Stepwise dissociation of histones from rat liver chromatin in alkaline solutions. Eur. J. Biochem., 43, 253-256. WIGGLESWORTH, V. B. 1964. The union of protein and nucleic acid in the living cell and its demonstration by osmium staining. Quart. J. Microsc. Sci., 105, 113-122. WORTHINGTON. 1972. Worthington Enzyme Manual, Worthington Biochemical Corporation, Freehold, New Jersey.

Nucleoprotein localization by bismuth staining.

TISSUE & CELL 1978 10 (2) 365-388 Published by Longman Group Ltd. Printed in Great Britain G. L. BROWN and M. LOCKE NUCLEOPROTEIN LOCALIZATION BISMU...
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