Variation in thin filament size in the skeletal muscle of the frog

ANDREA CORSI, ANNA LUISA GRANATA, CESARE VECCHI, ROSANNA STRABBIOLI

Laboratory of General Pathology, University of Ancona, 60100 Ancona, Italy

and LAMBERTO RE

Laboratory of Pharmacology, University of Ancona, 60100 Ancona, Italy

Summary

The sartorius muscle of Rana esculenta was fixedand dehydrated by different methods and cross-sections of sarcomeres of different lengths wereexamined by electron microscopy. The areawithin the outlines of the thin filaments in nega-tives was measured using a high-resolution tele-vision system and a graphics tablet or by using amicrodensitometer and computer processing theimages with a density threshold above the back-ground level.

In all specimens the area of the thin filaments in

the I-bands was found to be larger than in the S-zones of the A-bands where the thick and thinfilaments interpenetrate. The difference was stat-istically significant and independent of the lengthof the sarcomere. The results are in agreementwith previous observations in glycerol-extractedfibres. It is suggested that the change in size of thethin filaments might be accounted for by someinteraction with the thick filaments.

Key words: actin filament, microdensitometry, skeletalmuscle.

Introduction

In spite of much progress in recent studies, there issome uncertainty about the detailed structure of thethin filaments in skeletal muscle. Most models agreethat the actin subunits are oriented approximately atright angles to the filament axis (Amos et al. 1982;Egelman & De Rosier, 1983; O'Brien et al. 1983;Trinick et al. 1986). However, in the original model ofMoore et al. (1970) and the more recent models ofFowler & Aebi (1983) and of Smith et al. (1984), thelong axis of the actin monomeres is nearly parallel tothe filament axis. Thus this aspect of thin filamentstructure is still controversial. The traditional 65-75 Adiameter assumed for the thin filament would supportthe latter class of models. However, several obser-vations (Egelman & DeRosier, 1983; Egelman & Pad-ron, 1984; Trinick et al. 1986) appear to indicate thatthe diameter of the thin filaments extends to about100 A (reviewed by Egelman, 1985).

Some of these discrepancies may have arisen fromthe nature of specimens and the methods of prep-aration. In most cases investigations have been carriedout on thin filaments isolated from muscle or syntheticfilaments of pure actin, or from the complex of actinJournal of Cell Science 90, 569-575 (1988)Printed in Great Britain @ The Company of Biologists Limited 1988

and tropomyosin and troponin or the complex of actinand myosin SI. Also the well-documented variability ofthe F-actin helical pitch may afford an explanation as towhy different models can seem equally plausible. Itwas shown by Hanson (1967) and subsequently byothers (Egelman et al. 1982) that the periodicity of thetwist of the F-actin helix is variable. Several obser-vations indicate that there may also be structuralchanges in the actin filaments related to complexformation with myosin SI (Wakabayashi & Toyo-shima, 1981; Prochniewicz-Nakayama et al. 1983;Taylor et al. 1984) or with troponin (Toyoshima &Wakabayashi, 1985) and that also the Ca ion concen-tration may affect interaction between the actin strands(Toyoshima & Wakabayashi, 1985).

It appears that three-dimensional images from elec-tron micrographs of synthetic actin filaments or ofisolated thin filaments are unlikely to supply completeinformation on the structure of the thin filaments in themuscle fibre, where changes may occur because of thepresence of the thick filaments. For example, even inrelaxed muscle there may be transient binding of somemyosin heads to actin (see Stewart & Kcnsler, 1986, fora detailed discussion of this point).

569

Guba (1968) reported that the size of the thinfilaments was not constant throughout the sarcomere:according to his measurements in cross-sections of therabbit psoas, the diameter of the thin filaments is about65 A in the A bands and about 80 A in the I bands.

Here we present further evidence that the size of thethin filaments is larger in the I-bands in comparisonwith the S-zones, i.e. the overlap region of the A-bandswhere the thick and thin filaments interpenetrate. Thedifference does not seem to be simply a consequence ofa lack of order in the I band.

Materials and methods

Electron microscopy

The source of material was the sartorius muscle of the frog(Rana esculenta). Muscles were first fixed in situ, in anaes-thetized animals, at room temperature with 2-5 % glutaralde-hyde in (H M-cacodylate buffer, pH 7-2, for 1 h. The settingof the leg was chosen in order to obtain different sarcomerelengths in the specimens. Fixation with glutaraldehyde wascompleted in isolated small bundles of fibres for an additionalhour. In most cases post-fixation was carried out withbuffered 1 % osmium tetroxide for 1 h (at room temperature)and 4% uranyl acetate in 50% ethanol for 1 h. For somespecimens, after treatment with glutaraldehyde and osmiumtetroxide, additional fixation was provided by 2% tannic acidin cacodylate buffer for 30min at room temperature.

The material was dehydrated in an ethanol series andembedded in Epon-Araldite (Fluka). For comparison, in oneexperiment after post-fixation with osmium tetroxide anduranyl acetate, the tissue was dehydrated with acetoneinstead of ethanol (Page & Huxley, 1963).

Sections 450-500 A thick were cut perpendicular to theaxis of the myofibrils (after examining the samples, thesections were embedded again along with the grids and cuttransversly so that the thickness could be measured in theelectron microscope). For most experiments, after post-fixation with osmium tetroxide and uranyl acetate, serialsections were cut and collected on Robertson multiple-slotcopper grids so that one myofibnl could be studied over half asarcomere length.

The sections were stained with aqueous lead citrate (Ven-able & Coggeshall, 1965).

The micrographs were recorded with a Philips EM301 or aPhilips CM 12 electron microscope operated at a voltage of80 kV and at a real magnification of x67 000 or X75 000,respectively. In order to assess the real magnification, cali-bration was carried out by taking pictures of a grating replicaor a stained catalase resolution standard (Polysciences) underthe same conditions.

Image analysis

The negatives were analysed either by direct measurement orby microdensitometry.

Direct measurements were carried out in serial cross-sections, after post-fixation with osmium tetroxide and uranylacetate, by the use of a Grundig Electronic high-resolutiontelevision system (monitor BG444 and TV camera SN76), a

graphics tablet and an Apple II Plus computer.For microdensitometry a Perkin-Elmer PDS 1010G instru-

ment was used after post-fixation with osmium tetroxide anduranyl acetate, or alternatively with osmium tetroxide andtannic acid. Images of filaments were digitized at intervals of20|Um (corresponding to 3 A on the original object) andprocessed using a VAX 11/750 computer. The area wasdefined from the number of the pixels within the contour ofthe image. In order to define the contour, one threshold 75 %above the background value was chosen for all the thinfilaments; it is important to note that the background valuewas the same in the I and A-band regions. Although this wasto some extent an arbitrary choice of threshold, it would notinfluence the relative diameters of filaments since it was thesame for all micrographs measured. The images of the thinfilaments in the S-zones and in the I-bands were comparedwithin a section, in the same fibre. In each fibre the 12 bestindividual images in the I-bands and as many in the S-zoneswere selected on the basis of their shape and delimitationfrom the background.

Statistical analysis

For the experiments with serial sections, the data werearranged according to a factorial design with a fixed factor,the bands, and a random factor, the observers, and with anapproximate proportionality of the numbers of measurementscarried out by the observers for each level. Mixed modelanalysis of variance (Scheffe', 1959, p. 261) of the naturallogarithms of the data was carried out in each of thehemisarcomeres.

The t-ttst was used for the results of the other obser-vations.

Results

In all specimens that we examined, the area of the thinfilaments sections in the I-bands was found to be largerthan in the A-bands. The difference was statisticallysignificant in all cases. The data obtained in serialsections using a television system (see Materials andmethods) are shown in Table 1. The schematic dia-gram in Fig. 1 shows the distribution of data along thesarcomeres: it appears that the diameter of the thinfilaments was constant within both the I-bands and theA-bands.

Differences between observers were often signifi-cant, but they did not interact with the differencesamong sections, except in the case of experiment B (inthis case we tested separately the results of eachobserver). Differences among bands were alwayshighly significant; the difference between the general Imean and the general A mean accounted for most ofthis effect, whereas the differences among the I bandswere not significant. Also the differences among the A-bands were not significant, with the exception of thehemisarcomere E, where the means of the A-bandswere significantly different (see Table 2 for the relativeweights of Fl and F3).

570 A. Corsi et al.

Table 1. Diameters (A) of thin filaments measured by two observers (C.V. and A.L.G.) independently in thesame micrographs

Hemisarcomeres(length, f*m)

Observers

ind

IIS

I

sII

sIII

ssI

ssssI

ss

C.V.

62-2 ±1-762-6 ±2-350-6 ±2-4

64-2 ±2-852-7 ±1-9

61-4±3-060-8 ±2-649-9 + 2-3

62-1 ±3-363-3 ±3-160-613-649-6 ±2-550-312-561-612-150-312-751-712-950-412-649-3 1 2-4

61-912-450-81 1-949-312-1

(43)(40)(43)

(71)(68)

(76)

(71)(79)

(68)(69)(74)(78)(81)

(49)(74)(75)(72)(83)

(40)(40)(39)

A.L.G

63-5 12-562-613-349-712-3

68-513-553-113-2

61-813-962-613-248-712-5

63-813-663-4 + 3-262-814-450-2 + 2-850-712-4

62-2 + 2-650-312-752-5 + 3-551-312-449-7 + 2-8

62-7 1 2-850-2 + 2-549-312-7

(43)(40)(43)

(54)(53)

(59)(60)(62)

(58)(52)(59)(64)(68)

(48)

(71)(70)(68)(78)

(36)(40)(39)

A(1-35)

B(1-20)

C(1-15)

D(1-15)

E(1-05)

F(1-00)

The hemisarcomercs and the values are the same as shown in the diagram (Fig. 1).Values are means + S.D. In parentheses are given the numbers of the measured filaments. The calculation of the diameters from the

measured areas was based on the assumption that the shape of the filament section is a circle although it is not exactly so in the actualpictures.

A

v\62-2

62-6 Z

50-6

64-2

C

61-4

60-

49-9

52-7

D

621

63-3

60-6

49-6

50-3

E

61-6

50-3

51-7

50-4

49-3

F

I61-9

50-8

49-3

Fig. 1. A schematic diagram to illustrate distribution ofdata along the sarcomeres, which were cut in serial cross-sections of controlled thickness (S indicates thesuperposition zone, T indicates the transition zone). Thesarcomeres are the same as in Table 1.

In one specimen dehydrated with acetone (sarco-mere length 2-4 fim) the size of thin filaments was77-7 ±0-5 A in the I-bands and 65-2 ±0-4 A in the A-bands (these were the average values for 50 filaments)(f = 1908, 1,98 d.f., P<0-001).

In the specimens examined with a microdensit-ometer the values calculated for the diameter of thethin filaments were: 108-3 ± 1-8 A in the I-bands and86-7 ±0-8 A in the S-zones after post-fixation withtannic acid (t= 11-75, 1,22 d.f., P < 0 0 0 1 ) ;100-7±2-lA and 77-8±l-6A, respectively, afterpost-fixation with uranyl acetate (/ = 9-02, 1,22 d.f.,P<0-001). The sarcomere length was 2-4/im in thespecimens post-fixed with tannic acid and 2-3 (im in thespecimens post-fixed with uranyl acetate.

Figs 2-4 show electron micrographs of specimensafter different fixation and dehydration protocols.Figs 5, 6 show computer-processed images from thedensitometry data.

Discussion

The results show consistently that the size of the thinfilaments was different in the I-bands and in the S-zones of the A-bands: in our hands it was significantly

Thin filament size in frog skeletal muscle 571

Table 2. Tests on the differences between bands

Fl

F2

F3

A B (C.V.)

353-08 850-87

(1.2) (M37)

0-10

— -~

For each hcmisarcomcrc arc given F-tests and relevantgeneral I mean and general /

B(A.L.G.

559-21(1,105)

-

—

: degrees of

Hemisarcomeres

) c198-62

(1,2)

0-004

(1,2)-

D

905-60(1,4)

3-23(2,4)

1-36

(1-4)

freedom (in parentheses) for\ mean (Fl) , differences among I-bands (F2), differences among A-

results of the independent observers arc given separately (see text, Results).

E

6115-78(1,4)

--

114-22(3,4)

F

1110-81(1,2)

--

10-47

(1,2)

the following effects: difference betweenbands (F3). Foir hcmisarcomcrc B the

Fig. 2. Micrograph of a cross-section of frog sartorius muscle. The sample was post-fixed with uranyl acetate anddehydrated in ethanol. X22S000. A, A-band; B, I-band.

f*

^JS€5t»Fig. 3. Micrograph of a cross-section of frog sartorius muscle. The sample was post-fixed with tannic acid and dehydratedin ethanol. X225 000. A, A-band; B, I-band.

572 A. Corsi et al.

Fig. 4. Micrograph of a cross-section of frog sartorius muscle. The sample was post-fixed with uranyl acetate anddehydrated in acetone. X225 000. A, A-band; B, I-band.

. T i

Fig. 5. Densitometric reconstruction of cross-sections of actin and myosin filaments. The sample was post-fixed withuranyl acetate and dehydrated in ethanol. A, A-band; B, I-band.

"\

i*

Fig. 6. Densitometric reconstruction of cross-sections of actin and myosin filaments. The sample was post-fixed with tannicacid and dehydrated in ethanol. A, A-band; B, I-band.

Thin filament size in frog skeletal muscle 573

larger in the former. The difference was independentof the length of the sarcomere. This is not likely to bean artefact, since it is seen in all specimens afterdifferent fixation protocols, after dehydration bothwith ethanol and with acetone, and by using twomethods of image analysis.

The present results are in agreement with previousobservations in glycerol-extracted fibres (Corsi et al.1984).

It is not surprising that absolute measurements varywith the methods of handling the material and ofanalysis of the micrographs. The choice of a thresholdfor data processing after densitometry is necessarily tosome extent arbitrary. Therefore, we will not ventureinto a discussion of absolute diameters.

It seems unlikely that the difference is simply aconsequence of a lack of order in the I-bands. Theimages of obliquely cut filaments were not included inthe present study. The values derived from the examin-ation of well-isolated, roughly circular images of fila-ments were very close to one another with a smallstandard error of the mean, even in the longer I-bandswhere the disorder is likely to be more remarkable.

Crimping of the filaments in the I-bands mightaccount for the increase in diameter; however, thiswould imply that the thin filament should be shorter inthe longer sarcomeres and it would be difficult toreconcile with the data of Page & Huxley (1963) thatshow that the thin filament length is fairly constantover a wide range of sarcomere lengths.

It appears that a less casual and irregular change inthe thin filaments might occur as a consequence ofsome interaction with the thick filaments.

It seems plausible to relate the change in diameter toa change in conformation of the thin filaments. Achange in conformation of F-actin as a consequence ofinteractions with myosin was indicated by severalobservations (Wakabayashi & Toyoshima, 1981; Pro-chniewicz-Nakayama et al. 1983; Taylor et al. 1984;Toyoshima & Wakabayashi, 1985). It is worth notingalso that the thick filaments may have a slightlydifferent structure in the presence of thin filaments(Stewart & Kensler, 1986).

The capacity to bind many glycolytic enzymes in theI-bands might play a role in the change of size of thethin filaments. Evidence has been provided that glyco-lytic enzymes bind to specific sites of the thin filamentsby interaction with actin, tropomyosin and troponin,and that the interaction is influenced by Ca ionconcentration and by structural changes in the actin-tropomyosin-troponin filaments (Clarke & Morton,1976; Stewart et al. 1979, 1980; Clarke et al. 1980;Walsh et al. 1980). Since binding is limited to the I-bands it appears that it is dependent also on thepresence of thick filaments. The amount of glycolyticprotein in muscle fibres and the stoichiometry of the

binding seem adequate to account for an appreciablylarger diameter of thin filaments in the I-bands.

We are especially grateful to Drs C. F. Shoenberg and M.Stewart for helpful criticism and suggestions. We also thankDr G. Pittella for expert assistance and advice for densi-tometry and mathematical processing of the data, and Pro-fessor A. Colombi for help and criticism in the analysis ofvariance.

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(Received 7 March 1988-Accepted 5 May 1988)

Thin filament size in frog skeletal muscle 575