Silver staining the chromosome scaffold

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Silver staining the chromosome scaffold

EARNSHAW, William C., LAEMMLI, Ulrich Karl

Abstract

Cytological silver-staining procedures reveal the presence of a “core” running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this “core” and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic “core” staining was unaffected. Control experiments suggested that the “core” does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific “core” staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.

EARNSHAW, William C., LAEMMLI, Ulrich Karl. Silver staining the chromosome scaffold.

Chromosoma , 1984, vol. 89, no. 3, p. 186-192

DOI : 10.1007/BF00294997

Available at:

http://archive-ouverte.unige.ch/unige:127861

Disclaimer: layout of this document may differ from the published version.

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Chromosoma (Berl) (1984) 89:186-192

CHROMOSOMA

9 Springer-Verlag t984

Silver staining the chromosome scaffold

William C. Earnshaw 1 and Ulrich K. Laemmli

Departments of Molecular Biology and Biochemistry, University of Geneva, Sciences II, 30 Quai Ernest-Ansermet,

CH-1211 Geneva 4, Switzerland; 1 present adress: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA

Abstract. Cytological silver-staining procedures reveal the presence of a "core" running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this "core" and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory.

When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic "core" staining was unaffected. Control experiments suggested that the "core"

does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific "core" staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.

Introduction

Recent evidence suggests that the 250-A. chromatin fiber (DuPraw 1965; Gall 1966; Ris 1966) is folded into loops containing roughly 50-110 kilobases (kb) of DNA both at metaphase (Paulson and Laemmli 1977; Marsden and Laemmli 1979) and during interphase (Cook and Brazell 1978; Ig6-Kemenes and Zachau 1978; Adolph 1980; Vogel- stein etal. 1980; Lebkowski and Laemmli 1982; Hyde 1982). One model for the organization of metaphase chromosomes proposes that the bases of these loops are linked together by nonhistone proteins, which form an axial structure or "scaffolding" (Marsden and Laemmli 1979;

Laemmli et al. 1978). This model is based on biochemical studies which showed that the metaphase DNA retains a compact structure even after complete extraction of the histories (Adolph et al. 1977a; Lewis and Laemmli 1982), and on two types of electron microscopic evidence. First, electron micrographs of histone-depleted chromosomes show a halo of DNA consisting of many loops surrounding a dense central region (Marsden and Laemmli 1979). Sec- ond, transverse thin sections through expanded chromosomes show a radial starlike arrangement of the chromatin fibers (Marsden and Laemmli 1979).

The metaphase scaffold consists of two major proteins, SC1 (Mr--- 170,000 daltons) and SC2 (M r = 135,000 daltons) and a number of minor proteins (Lewis and Laemmli 1982).

Metalloprotein interactions are required for the mainte-

nance of scaffold structure. The metal involved is either Cu 2 + or Ca 2 +; its chelation leads to dissociation of the scaffold and to complete unfolding of the DNA (Lewis and Laemmli 1982). The isolated scaffolding is structurally stable under certain conditions despite extensive prior digestion with nucleases and extraction of proteins (Adolph et al.

1977a). Electron microscopic examination shows it to be composed of a loose fibrous network, which retains the metaphase shape and appears to have a residual kineto- chore structure (Adolph etal. 1977b; Earnshaw and Laemmli 1983).

It is usually impossible to observe the scaffolding in intact chromosomes either when they are spread for electron microscopy (Earnshaw and Laemmli 1983; Rattner et al. 1975; Labhart et al. 1982) or in thin sections (Marsden and Laemmli 1979; Okada and Comings 1980). This is not surprising since the fibrous scaffold network is not easily distinguished from chromatin fibers (Earnshaw and Laemmli t983) and since the total mass represented by the scaffold is < 2% of the total chromosome mass (Lewis and Laemmli 1982). To visualize the scaffold proteins in intact chromosomes a selective staining method is required.

Electron microscopy of whole-mount preparations of meiotic pachytene chromosomes suggests that the chromatin fiber is organized into loops which attach to the lateral elements of the synaptonemal complex (Moses 1956; Com- ings and Okada 1972; Rattner et al. 1980a, b). The synaptonemal complex has recently been shown by a number of groups to selectively bind silver (Pathak and Hsu 1979;

Fletcher 1979; Dresser and Moses 1979), and this suggested that silver might also selectively stain the scaffolding in intact mitotic chromosomes. In fact, silver does stain an axial core-like structure in mitotic chromosomes (Howell and Hsu 1979; Satya-Prakash et al. 1980; Burkholder and Kaiserman 1982; Zheng and Burkholder 1982). Some studies suggest that the axial "core" may be composed of nonhistone proteins (Howell and Hsu 1979) of the chromosome scaffolding (Adolph etal. 1977a; Lewis and Laemmli 1982), but others suggest that the staining is due to nonspecific aggregation of chromatin (Zheng and Burkholder 1982; Burkholder 1982).

This communication deals with the relationship between the axial "core" stained with silver in intact metaphase chromosomes and the chromosome scaffold isolated and characterized previously (Lewis and Laemmli 1982; Adolph et al. 1977b; Earnshaw and Laemmli 1983). We have used in situ treatments of unfixed chromosomes adsorbed to

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Fig. 1 a-d. Effects of various treatments on silver staining of the chromosome core. a Chromosomes pretreated with RSB buffer, b Chromosomes digested in situ with 25 gg/ml micrococcal nuclease prior to silver staining, e Chromosomes pretreated in situ with NaC1 lysis mix and subsequently digested with 25 btg/ml micrococcal nuclease. This treatment worked equally well when the order of these steps was reversed. Note the removal of the peripheral chromatin halo in b and e. d Chromosomes pretreated with 100 gg/ml pronase prior to silver staining. No axial core is seen. The material which remains is rich in DNA as indicated by DAPI staining.

Bar represents 10 gm

slides, and electron microscopy of isolated scaffolds to show that components of the axial "core" observed when intact chromosomes are stained with silver are also components of isolated scaffolds. The data suggest that the scaffold is the primary target of the silver stain in chromosomes.

(1982) and stored at 0~ in RSB buffer plus 0.1% digitonin. Scaffolds were isolated using the dextran sulfate/he- parin procedure (Paulson and Laemmli 1977), and prepared for electron microscopy by a gentle sedimentation technique (Earnshaw and Laemmli 1983).

Materials and methods

Enzymes were obtained from Millipore; DAPI (4',6-diami- dino-2-phenylindol-dihydrochloride), from Boehringer;

and Trasylol (Aprotinin), from Mobay Chemical Co., New York.

RSB buffer contains 10 mM Tris:HC1 pH 7.4, 10 mM NaC1, and 5 mM MgC12. High salt lysis mix contains 20 mM Tris HC1 pH 9, 20 mM Na-EDTA (ethylenedi- aminetetraacetic acid) pH 9, 0.2% Ammonyx Lo, and 4 M NaC1.

Chromosome and scaffold isolation. Chromosomes were isolated by the polyamine method of Lewis and Laemmli

Silver staining. A 5-~tl sample of chromosome solution (opti- cal density 260=5-10) in RSB plus 0.1% digitonin was spotted onto a clean slide and spread with the pipette tip to cover a diameter of 1-2 cm. The slide was dried for from 1 h to overnight at room temperature. For enzyme treatments, several drops of enzyme (with Trasylol freshly added to 10 KIU/ml) were placed over the dried chromosomes, covered with a coverslip, and incubated on an ice- cold metal block for 20 min. The slide was then rinsed with water and dried again at room temperature. Pretreat- ment with 50% AgNO a was done the same way except that the block was heated to 37 ~ C (for 20 min), 47 ~ C (for 5 min), 57~ (for 5 min), or 67~ (for 5 min). The stan- dard treatment was 67 ~ C for 5 min.

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Fig. 2a--d. Silver and fluorescence staining of isolated chromosomes, a Untreated chromosomes stained with silver, b Fluorescence microscopy of the field shown in a. Staining is with the DNA-binding dye DAPI. e Chromosomes digested in situ with 25 gg/ml micrococcal nuclease. Nuclease-treated chromosomes have "naked" cores, whereas untreated chromosomes have a halo around the core. d DAPI fluorescence of the field shown in e. Residual DNA is undetectable by this method. Bar represents 10 gm

Silver staining was performed using the Ag-AS method of Bloom and Goodpasture (1976), with the following mod- ification suggested by Dr. M. Jotterand-Bellomo. During acidification of the formalin developer, formic acid was added dropwise until a mixture of one drop developer and one drop ammoniacal silver (AS) solution took 75 s before showing visible precipitate. For staining, two drops each of developer and AS solution were mixed in a 1.5-ml plastic tube. A small drop of mixture was quickly spotted onto a pretreated area of chromosomes on a slide and covered with a coverslip, and the staining reaction was followed in the microscope. When the desired degree of staining was observed, the reaction was halted by rinsing exhaustively with water. For electron microscopy, chromosomes or scaffolds were sedimented onto carbon-coated gold grids. Pre- treatment and staining were accomplished by submerging the grid below a coverslip on a slide and proceeding as for chromosomes on slides. The progress of staining was monitored in the light microscope by direct observation of the structures on the carbon film.

Results

Axial core elements can be visualized by pretreating methanol:acetic acid-fixed mitotic chromosomes with either NaOH or HC1, or by prolonged hypotony followed by staining with silver nitrate (Howell and Hsu 1979; Satya-

Prakash et al. 1980; Burkholder and Kaiserman 1982). The 3 : 1 methanol: acetic acid treatment, commonly used in cytological preparations, is known to extract some chromosomal proteins (Comings 1978; Burkholder and Duczek 1980). We wished to know if "cores" could be observed in purified, unfixed chromosomes after silver staining. In these chromosomes the differential enhancement of the axial "core" versus the peripheral chromatin was obvious (Fig. 1 a). This staining could be observed after pretreatment of the chromosomes with 50% AgNO 3 at tempera- tures as low as 37 ~ C. Other pretreatments were unneces- sary.

We have used unfixed chromosomes to extend previous experiments that suggested that the "core" must be largely composed of nonhistone proteins (Howell and Hsu 1979).

If chromosomes adsorbed to slides were digested with micrococcal nuclease prior to silver staining, the "core" staining was unaffected (Fig. 1 b), but the peripheral chromatin halo was removed (particularly visible by comparing chromosome clumps in Fig. I a, b). Combining nuclease digestion with high-salt extraction also had no effect on the observed staining (Fig. 1 c). The combination of nuclease and high-salt treatment is known to extract at least 99% of both histones and DNA and to expose the residual scaffold (Adolph et al. 1977a). To verify that the nuclease treatment effectively removes the bulk of the DNA when chromosomes are digested in situ, we poststained the above slides

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Fig. 3a, b. Electron microscopy of silver-stained chromosomes and isolated scaffolds, a An intact chromosome lightly stained with silver. Selective staining of the kinetochores is observed, b Isolated residual scaffolds stained with silver. These scaffolds appear substantially smaller than the chromosome shown in a, possibly due to the high ionic strength of the silver pretreatment (Earnshaw and Laemmli 1983). Bar represents 1 gm

with DAPI, which fluoresces when bound to DNA (Wil- liamson and Fennel 1975; Morikawa and Yanagida 1981), and examined them in the fluorescence microscope. Figure 2a, b shows the axial "core" and the peripheral chromatin ofunextracted chromosomes by bright-field and by fluorescence microscopy. The bright fluorescence of the peripheral halo in panel (b) indicates that the chromosomal DNA surrounds the axial structures. The central axis is dark, indicating little DNA in this area (or possibly obscurement of the fluorescence by the local high concentration of silver).

Exposure of the chromosomes to nuclease prior to staining removes the fluorescent halo but not the central axis (Fig. 2c, d). Thus, removal of most of the chromatin prior to silver staining does not reduce the staining of the axial structure.

The axially stained material appears to be predominantly composed of proteins (Howell and Hsu 1979). Treatment of unfixed chromosomes adsorbed to slides with pronase

(100 ~tg/ml) eliminates the axial staining (Fig. I d). The re- sultant weakly staining puddles give rise to an intense, even fluorescence when poststained with DAPI, indicating that they are rich in DNA (not shown). The axial structure does not require the presence of RNA in a form accessible to enzymes since pretreatment with RNase A (50 I~g/ml) has no effect on silver staining of the chromosome axis (not shown).

Extraction procedures carried out on glass slides are difficult to monitor biochemically. It was important to show that scaffolds isolated by solution techniques (Lewis and Laemmli 1982) would also show a specific binding affinity for silver. Therefore scaffolds were isolated in vitro, sedimented onto carbon-coated grids (Earnshaw and Laemmli 1983), stained with silver nitrate, and examined in the electron microscope. In this procedure the same preparation of scaffolds is monitored by both SDS polyacrylamide gel electrophoresis and electron microscopy.

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Figure 3a shows an electron micrograph of a silver- stained intact chromosome. Note the staining of the axial

"core" and accentuation of the kinetochores. When isolated scaffolds were treated under identical conditions, they were heavily stained with silver (Fig. 3b). The axial fiber of isolated scaffolds appears more compacted than its coun- terpart in intact chromosomes, probably due to contraction induced by the high ionic strength of the silver nitrate solution. We have previously shown that the appearance of the scaffolding is highly dependent on the ionic strength.

A loose, expanded structure is seen at low ionic strengths, whereas a compact structure is observed at higher ionic strengths (Adolph et al. 1977b; Earnshaw and Laemmli 1983).

Discussion

In order for meiotic chromosomes to pair correctly, the homologues must apparently be aligned by a proteinaceous scaffold, the synaptonemal complex (Moses 1956; von Wettstein 1971), which may be observed directly in both thin sections and whole mounts of meiotic chromosomes (Westergaard and yon Wettstein 1970; reviewed in Comings and Okada 1972). It may also be observed by light microscopy if the chromosomes are stained with silver (Pathak and Hsu 1979; Fletcher 1979; Dresser and Moses 1979).

Recent evidence suggests that mitotic chromosomes may also be organized by a scaffolding structure (Paulson and Laemmli 1977; Adolph et al. 1977a, b; Lewis and Laemmli 1982). However, this structure is much more diffuse than the synaptonemal complex and consequently cannot be observed directly in intact chromosomes. As is the case for the synaptonemal complex, an axial structure can be made visible by staining acid-fixed mitotic chromosomes with silver nitrate following pretreatment with NaOH or HC1, or by prolonged hypotony (Howell and Hsu 1979; Satya- Prakash et al. 1980). Our experiments show that such pretreatments are not necessary. We have observed axial staining when highly purified unfixed chromosomes are directly stained by silver nitrate. It is unclear why unfixed chromosomes need no pretreatment to allow axial staining, al- though they may disperse more easily to allow differential staining of the peripheral chromatin versus the axial structure.

If chromosomes adsorbed to slides are extensively digested with DNase or RNase, extracted with 2 M NaC1, or subjected to combinations of these treatments, the

"core" material stainable with silver remains. Therefore, the" core" is likely to contain little DNA, RNA, or histone, in accordance with previous biochemical characterizations of isolated residual scaffolds (Adolph et al. 1977a; Lewis and Laemmli 1982). This conclusion is supported by the demonstration that scaffolds isolated in vitro can be intensely stained with silver nitrate under conditions that stain the "core" in intact chromosomes. These data suggest that the axial structure made visible by silver staining corre- sponds to the chromosomal scaffolding.

If chromosomes are exposed to shear, it is possible to observe the formation of corelike substructures by light and electron microscopy. These structures have been termed

"core fibers" (Stubblefield and Wray 1971) or" unit fibers"

(Baket al. 1977). They are distinct from the nonhistone scaffold described above in that they contain substantial

amounts of DNA and histone, components that are quanti- tatively removed during scaffold isolation (Adolph et al.

1977b; Lewis and Laemmli 1981). We have shown that in some cases these corelike structures arise from chromosomes by a twisting process similar to the spinning of yarn (Earnshaw and Laemmli 1983; Earnshaw, in preparation) and not by the stripping away of peripheral material (Stubblefield and Wray 1971). We, therefore, feel that these structures play no role in the silver staining described in this report.

I~ has also been reported that the chromosome core contains a bundle of axially oriented DNA fibers, observed when the chromosomes have been prepared for microscopy after exposure to shear (Stubblefield and Wray 1971) or after being stretched during surface spreading procedures (Mullinger and Johnson 1980). In these experiments parallel axial DNA fibers could arise from stretching of radially looped chromatin fibers, as occurs during double loop bridge formation when lampbrush chromosomes are stretched (see Callan 1981). We know of no published report where such bundles of parallel DNA fibers have been observed on the chromatid axis of unstretched chromosomes. At any rate, the scaffold as we isolate it contains little DNA detectable by either use of isotopic labeling (Lewis and Laemmli 1981), electron microscopy (Earnshaw and Laemmli 1983), or by fluorescence staining with DAPI (this report). Our experiments suggest no essential role for DNA in the silver staining described above.

It is unlikely that the "core" is composed of nucleolar components, even though these are known to stain with silver (Goodpasture and Bloom 1975; Howell et al. 1975), since preineubation of chromosomes with RNase fails to abolish the specific "core" staining (Howell and Hsu 1979;

this study). We expected this result since during our chromosome isolation procedure (Lewis and Laemmli 1982) the chromosomes are exposed to RNase to minimize adherence of cytoplasmic components. Also silver-stained nucleolar components are found predominantly on the surface of metaphase chromosomes (Schwarzacher et al. 1978; Pawe- letz and Risueno 1982) and not along the axis.

It has recently been suggested that the "core" staining merely reflects the greater amount of protein mass along the chromatid axes (Zheng and Burkholder 1982; Burk- holder 1982). This is unlikely to explain the silver-staining results for three reasons. First, when chromosomes are stained with a general amino-reactive compound, dansyl chloride, a diffuse homogeneous staining is observed indicating that the bulk of the histone mass is not concentrated on the chromosome axis (Utakoji and Matsukuma 1974;

Matsukuma and Utakoji 1976). Burkholder (1982) used this same stain, but previously subjected the chromosomes to harsh pretreatments and BrdU substitution. Second, when chromosomes are stained with silver and the DNA subsequently detected by fluorescent staining with DAPI (Fig. 2b), the chromatin is found to be evenly dispersed on the slide after silver staining. No evidence of a greater concentration of DNA (chromatin) along the chromosome axis was obtained. Third, isolated scaffolds, in which histones are undetectable in SDS polyacrylamide gels and which contain only about 2% DNA by weight (Earnshaw and Laemmli 1983), stain intensely with silver. This argues against the staining of whole chromosomes arising from a nonspecific interaction of silver with either histone or DNA.

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191 Our experiments suggest that the axial "core" detected

in mitotic chromosomes by the silver-staining technique is composed of a set of nonhistone proteins distributed along the chromatid axis that is specifically enhanced by the silver-staining procedure. Some of these proteins are asso- ciated with the previously characterized chromosome scaffolding (Adolph et al. 1977a, b; Lewis and Laemmli 1982;

Earnshaw and Laemmli 1983).

Acknowledgements. We are grateful for advice and protocols re- garding the silver-staining technique from Dr. M. Jotterand-Bel- lomo, Centre Hospitalier Universitaire Vaudois, Lausanne, and for the suggestion to use gold grids from Dr. J. Kistler. The work was supported by the Swiss National Science Foundation grant 3.621.80, and by the canton of Geneva.

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Received August 22, 1983 / in revised form November 9, 1983 Accepted by T.H. Hsu