J. Cell Sci. 23, 43-55 (1977) 43

Printed in Great Britain

MITOTIC SPINDLES OF DROSOPHILA

MELANOGASTER: A PHASE-CONTRAST AND

SCANNING ELECTRON-MICROSCOPE STUDY

AMY MILSTED* AND WILLIAM D. COHEN

Department of Biological Sciences, Hunter College of The City University of NeivYork, New York, New York 10021, U.S.A.

AND NINA LAMPEN

Memorial Sloan-Kettering Cancer Center, 410 East 6Sth Street, New York,Netv York 10021, U.S.A.

SUMMARY

Mitotic spindles have been isolated from the blastema stage of Drosophila melanogasterembryos using modified tubulin-polymerizing medium. 'Clean' spindles, relatively free ofcontaminating cytoplasmic material, are obtained. Under phase contrast, mitotic stages appearremarkably similar to those seen in situ, as reported in early literature. This preservation ofmorphological integrity, coupled with relative structural simplicity due to low chromosomenumber (zn = 8), makes these spindles ideal subjects for study. Use of the scanning electronmicroscope provides excellent visulization of their general structural organization, changes inwhole spindle structure during the course of mitosis, and higher resolution viewing of surfacedetail than is permitted with light microscopy.

INTRODUCTION

The structure of isolated mitotic and meiotic spindles has been examined previouslyby a variety of techniques, including thin sectioning (Kane, 1962; Goldman &Rebhun, 1969 ; Cohen & Gottlieb, 1971) and negative staining (Kiefer, Sakai, Solari &Mazia, 1966) for transmission electron microscopy (TEM), high voltage TEM(Mclntosh, Cande, Snyder & Vanderslice, 1975), phase-contrast, polarization andinterference light microscopy (Kane & Forer, 1965 ; Rebhun & Sander, 1967 ; Cohen,1968 ; Forer & Goldman, 1972 ; Inoue, Borisy & Kiehart, 1974).

We describe here a study of isolated mitotic spindles of Drosophila melanogasterutilizing the scanning electron microscope (SEM). With the great depth of field and3-dimensional imaging of objects which it can give, the SEM is superior to other typesof microscope for visualization of general structural organization. In usual applica-tions, with specimens such as whole cells or pieces of tissue, internal structure cannotbe seen. Recently, however, it has become apparent that the SEM is applicable to thestudy of organelles, with greatest value in the case of structures too large or thick forTEM whole mounts (Kersey & Wessells, 1976 ; Kirschner, Rusli & Martin, 1975).The isolated mitotic spindle falls in this category; with appropriate preparation

• Present address: Carnegie-Mellon University, Department of Biological Sciences,Pittsburgh, Pennsylvania, 15213, U.S.A.

44 A. Milsted, W. D. Cohen and N. Lampen

methods there will be no surrounding membrane or other material obscuring the viewof its fibrous structure.

Drosophila melanogaster mitotic spindles isolated at the blastema stages appear underphase contrast to have relatively simple structure (Milsted & Cohen, 1973). With theirlow chromosome number (zn = 8) and obvious fibres they seemed a promisingsubject for study with the SEM. In the course of this work we found that cleanspindles, free of most adhering cytoplasmic material, could be prepared by using amodified tubulin polymerization medium (Rebhun, Rosenbaum, Lefebvre & Smith,1974). In such specimens spindle and astral fibres, chromosomes, and a complexstructure in the polar region can be seen. Phase-contrast light microscopy and scanningelectron microscopy together allow characterization of major changes in spindleorganization during the mitotic cycle in these embryos.

MATERIALS AND METHODS

Wild-type Drosophila melanogaster were obtained from the Carolina Biological Supply Co.(Burlington, North Carolina, U.S.A.) and were cultured in 'Instant Drosophila Medium'(Carolina). Embryos were collected and dechorionated as described previously (Milsted &Cohen, 1973) and allowed to develop to the blastema stage during which hundreds of nucleiundergo synchronous syncytial mitosis (Sonnenblick, 1950). Isolated spindles can be preparedat this stage using hexylene glycol medium (Milsted & Cohen, 1973). However, better prepara-tions for microscopy and for physiological experiments can be obtained using modified micro-tubule polymerization medium (TPM) based on that of Rebhun et al. (1974). The T P Mcontained o-i M PIPES (piperazine-iV-iV'-bis-[2-ethane sulphonic acid]), 1 mM MgCl,, 5 mMEGTA (ethyleneglycol-bis-[-aminoethyl ether] JV,iV'-tetraacetic acid), 10 mM TAME (p-tosylarginine methylester HC1), 1 mM GTP (guanosine s'-triphosphate) when used, and 0 4 %Triton X-100, brought to pH 6-8 with KOH.

Preparations intended only for phase-contrast microscopy were made as follows : one or moredechorionated embryos of the appropriate stage were transferred from the culture dish to aslide, in a drop of water. The water was totally removed (embryos do not dehydrate easily) andreplaced by a small drop of T P M containing GTP. A clean No. 1 coverslip was added, and itsweight was generally sufficient to rupture the vitelline membrane of the embryo, allowing thespindles to spill out and spread between coverslip and slide. In some cases, lysis was induced bygentle tapping of the coverslip. The term 'isolated' is thus applied to the spindles in suchpreparations in its broad sense, indicating physical separation of spindles from cytoplasm andsubstitution of an experimental medium for the in vivo environment.

Observations were made using a Zeiss phase-contrast microscope (oil immersion lens N.A.1 3) and spindles were photographed on Kodak Plus-X 35-mm film. Glass coverslips (as opposedto plastic ones used for SEM) permitted the clearest viewing and photography.

For routine preparations, the embryos were cultured at room temperature and lysed in T P Mat room temperature (21-22 °C). Additional experiments were performed in which the de-chorionated embryos were cooled prior to lysis, in attempts to obtain spindle polar regions inwhich the microtubule organizing centre ( ' M T O C : Pickett-Heaps, 1969) might be seen moreeasily. Some success was achieved by chilling for approximately 25 min at 6 °C, followed bylysis in TPM without GTP at room temperature.

For SEM, plastic coverslips were employed (A. H. Thomas Co., Philadelphia, Pa., U.S.A.).These were pre-cleaned for a minimum of 1 h in dilute 'Micro' cleaning solution (Int. Prod.Corp., Trenton, N.J.), rinsed with water, and incubated in a solution of polylysine in water at1 mg/ml (Mazia, Schatten & Sale, 1975) for up to 48 h, under refrigeration to prevent bacterialcontamination. Subsequently the coverslips were rinsed thoroughly in water and allowed to dry.Glass slides were silicone coated (Siliclad, Clay Adams, Inc., N.Y., N.Y.) in order to favouradhesion of the spindles to the polylysine-coated coverslips. Embryos in the 9th to 12th divisionspost-fertilization were lysed in TPM without GTP, and the spindles perfused with additional

Drosophila spindles in phase contrast and SEM 45

T P M to remove much of the debris. Within 5 min of lysis, T P M containing 2 0 or 25 % glu-taraldehyde at pH 6-8 was perfused under the coverslip, and the preparation, still on the slide,was placed in a moist chamber for 1-1-5 h. The chamber consisted of a Petri dish with moistfilter paper in the bottom. Glutaraldehyde was removed by perfusion with several changes ofo-i M PIPES buffer, pH 6-8. Subsequently 1 % OsO4 ino-i M PIPES, pH 6 8 , was perfusedunder the coverslip and allowed to remain there for 30—60 min, with the preparation again heldin the moist chamber. Osmium was then removed by perfusion with several changes of buffer.

Preparations were examined under phase contrast for possible candidate spindles suitable forfurther SEM processing. In general, the best specimens were those lying alone, free of otherspindles and adhering debris. That there were relatively few such spindles remaining is probablydue to perfusion, during which spindles occasionally are seen to detach from the coverslips.

The pre-selected coverslip preparations were carefully removed from their slides and placedin small Petri dishes of 0 1 M PIPES, pH 6-8. They were then dehydrated in an ethanol seriesand passed through a graded series of ethanol-Freon 113 mixtures into 100% Freon 113(CCljF-CCljF). Critical point drying (Anderson, 1951) was carried out in Freon 13 (CC1F3)(Cohen, Marlow & Garner, 1968).

Critical-point-dried specimens were examined, while dry, under phase contrast. Spindleswere still recognizable, and they were photographed for later reference and their locationmarked by circling or scratching the region with a Scheaff micro-object marker (A. H. ThomasCo., Philadelphia, Pa., U.S.A.) mounted on the microscope. Appropriate areas of the plasticcoverslips were cut out with scissors and mounted on SEM stubs with double-sticky tape.Plastic coverslips were used for all SEM preparations because they could be marked andtrimmed easily.

Silver conductive paint was applied to the coverslip edges on the stub, and the preparationwas coated with approximately 20 run of gold or gold-palladium in a Denton rotating, tiltingvacuum evaporator. Specimens were examined and photographed in a Cambridge Stereo-scan S4 SEM at 20 kV, using zero tilt for easiest recognition of preselected individual spindleswhen compared with light micrographs (phase contrast).

RESULTS

Phase-contrast observations

When viewed under phase contrast, blastema stage spindles prepared in TPM areeasy to see and usually free of adhering material. Occasionally they are clumpedtogether in masses, but in most preparations there are many individual spindles whichcan be examined. Spindle fibres, chromosomes, midbodies, and rather large structuresat the expected location of centrioles are obvious. In any one preparation, all of thespindles are in approximately the same mitotic stage, as expected. By slight alterationsin the timing of embryo lysis, most of the major mitotic stages can be obtained indifferent preparations. These are illustrated in Figs. 1 to 5, with mitotic stagesdesignated according to Huettner (1933).

In Fig. 1, the triangular morphology typical of the Drosophila melanogaster blastemastage prophase spindle is apparent. Black spots with fibrous material radiating fromthem appear at the 2 presumptive polar positions (c and c'). The third corner of thetriangle lacks this feature and is more rounded, so that the entire structure is sac-like.The chromosomes (ch) are grouped in the midregion and seem to be organized into2 subgroups, each of which has a distinct focal point (arrows) located midway alongthe line between c and c .

The polar structures are about 0-7 /mi in diameter as measured in Fig. 1 and in allother spindle preparations. In phase contrast they are always seen in prophasespindles, usually in metaphase, and less frequently in later stages. Since their size is

4 CEL 23

A. Milsted, W. D. Cohen and N. Lampen

,<<*

• - c '

Drosophila spindles in phase contrast and SEM 47

greater than that expected for a single centriole or centriole pair, we have tentativelyassigned to them the term 'centriole complex'.

At metaphase (Fig. 2) the spindle poles are well denned by centriole complexes. Asmall number of major fibres, generally three or four, are evident between chromo-somes and poles. The fibres seem to converge at a point a short distance from thecentriole complex, where they coalesce into a constricted neck region (x, also visible inHuettner's (1933) spindles in situ) which apparently connects directly to the centriolecomplex. Chromosomes (ch) are seen positioned in the spindle mid-plane.

Figs. 3 and 4 show typical late anaphase spindles. There are a small number ofmajor interzonal fibres, probably four, each of which is thickened in the midregionforming an apparent midbody. In Fig. 3 a mass of spindles is seen; even in suchmasses structural details such as midbodies (m) and apparent centriole complexes canoften be detected with careful focusing. As this figure shows, synchrony in any onepreparation is not absolutely perfect: while most spindles were in anaphase (a), a fewhad entered telophase (f). In the spindle shown in Fig. 4, four midbodies are visible(m). Here the chromosomes are massed at the poles except for a trailing arm in eachchromosome set (x). The arms are identifiable as such on the basis of careful focusingthrough several planes, which shows them to be continuous with the polar chromatinand considerably thicker than the interzonal fibres. Such trailing arms were seen byRabinowitz (1941) in situ, and we have confirmed their presence by thin-sectioning(Cohen & Milsted, unpublished observations). As seen in phase contrast, these arms

Fig. 1. A group of prophase spindles, in isolation medium, c, c, ' centriole complexes' ;ch, chromosomes ; arrows indicate chromosome sub-group focal points. Phase contrast,x 1740.

Fig. 2. A metaphase spindle, in isolation medium ; c, c', centriole complexes, ch,chromosomes ; x, constricted region at fibre convergence point, apparently connectingdirectly to centriole complex. Phase contrast, x 2060.

Fig. 3. Spindles trapped in a mass. Most of them were in late anaphase, such as spindle(a); one is in telophase, spindle (t); c, presumed centriole complexes ; m, midbody.Phase contrast, x 2060.

Fig. 4. A late anaphase spindle, in isolation medium ; ch, chromosomes massed nearpoles ; if, interzonal fibre ; m, one of 4 adjacent midbodies ; x, trailing chromosomearm (one in each half-spindle). Phase contrast, x 2060.

Fig. 5. A telophase spindle, typically bent at this stage, in isolation medium, m, singlemidbody. Phase contrast, x 940.

Fig. 6. Spindle polar region obtained by lysis of precooled embryo in TPM ; centralcentriole complex is present. Phase contrast, x 1740.

Fig. 7. Three early prophase spindles, isolated in TPM and fixed, dehydrated, andcritical-point dried on polylysine-coated coverslip, as described in Materials andmethods ; c, c , apparent focal points for fibrils, although polar structure is not visible ;X, crossed scratch lines on coverslip ; Y, another scratch line ; 1, 2, 3, particles oncoverslip (scratches and particles used as landmarks for comparison with Fig. 8).Scanning EM, x 1400.

Fig. 8. The same 3 early prophase spindles shown in Fig. 7, as seen under phasecontrast after critical-point drying, prior to gold coating. Note landmarks correspond-ing to those in Fig. 7 : X, crossed scratch lines on coverslip ; Y, another scratch line ;1, 2, 3, particles on coverslip ; c, c\ apparent focal points. Phase contrast, x 940.

4-2

A. Milsted, W. D. Cohen and N. Lampen

Drosophila spindles in phase contrast and SEM 49

extend toward the same midbody in each half-spindle, as if associated with the sameinterzonal fibre.

Even telophase spindles can be isolated (Fig. 3, spindle (t); Fig. 5). This stage isdistinguished from late anaphase by the fact that there is but one relatively thickinterzonal fibre with a single large midbody (m), and by the reconstituting daughternuclei at the polar extremities. The entire structure is usually curved or bent.

If embryos are cooled prior to lysis, it is possible to obtain preparations withincomplete spindles and small, sometimes separate polar regions, in which the centriolecomplex is seen to advantage (Fig. 6). A number of 'points' radiate from the blackcentral structure, the diameter of which is in the range 0-7-0-8 /tm.

Scanning EM observations

In the description which follows, the term 'fibril' is used to indicate the relativelythin structures seen in the SEM, and the term 'fibre' is applied to obvious bundles offibrils.

Early prophase spindles, as seen in the SEM (Fig. 7), all present the appearance ofmasses of tangled fibrils. In one of the spindles shown, 2 major focal points for thefibrils are apparent (c and c'). As seen at higher magnification in Fig. 9, no majorfibres are evident, and chromosomes are not visible at this stage using the SEM. Forcomparison, Fig. 8 shows the same 3 spindles as Fig. 7, after critical point drying andprior to coating, under phase contrast. They are recognizable as such both by theirrelative positions and by the particles and scratches which correspond (for example,particles numbered /, 2, 3, crossed scratch lines labelled X, a scratch labelled Y, inboth Figs. 7 and 8). Since the spindles are viewed dry at this point, not much detail isseen in phase contrast, but the light micrograph is valuable in locating good specimensfor subsequent scanning.

In late prophase spindles such as that in Fig. 10, relatively thick, elongate smooth-

Fig. 9. Higher magnification view of the prophase spindle in upper right corner ofFig. 7 ; /, fine fibrils, in approximately parallel array. Chromosomes are not visible atthis stage in the SEM. Scanning EM, x 5830.Fig. 10. A late prophase spindle, somewhat flattened on coverslip ; ch, chromosomes ;crossed fibrils at arrow. Scanning EM, x 5200.Fig. 11. A late anaphase spindle, showing fibres composed of thinner fibrils, af,astral fibrils ;/, central fibre. Scanning EM, x 4170. Inset: The same spindle, as seen inphase contrast after critical-point drying, prior to gold coating. Highly refractile areascorrespond to regions of greatest density. Phase contrast, X 785.Fig. 12. Higher magnification of the lower polar region of the spindle shown inFig. 11 : cc, centriole complex; ch, chromosome arms ; / , a thin fibril on coverslipsurface ; p, a point at periphery of centriole complex, to which 3 very fine fibrils areconnected. Scanning EM, x 8330.Fig. 13. A telophase spindle, with curved structure ; interzonal fibril bundle seemstwisted, as does path of fibril (/) ; n, daughter nuclei at spindle extremities. ScanningEM, x 5200.Fig. 14. Higher magnification of one of the daughter nuclei in the telophase spindleshown in Fig. 13 ; r, ring-like surface features. Scanning EM, x 9830.

SO A . Milsted, W. D. Cohen and N. Lampen

surfaced structures are found in the position expected for chromosomes on the basis ofphase-contrast observations of the same preparations. Based upon their thickness range(0-3-0-4 /<m) and location, we believe these to be the chromosomes and have desig-nated them as such (ch). At this stage the number of individual fibrils seems relativelysmall, and they are not yet organized into a recognizable spindle with major fibresand distinct poles. Apparent multiple focal points for the fibrils give the spindleend-region a criss-crossed appearance (arrow).

A late anaphase spindle is shown in Fig. 11. Here the poles are well defined byconvergence of fibrils. In the midregion the spindle is composed of a relatively smallnumber of interzonal fibres, each consisting of smaller fibrils. The central fibre (/) isthicker than the others, measuring about 0-7 /tm in diameter, with component fibrilsin the range of 0-1-0-2 /tm. Apparent astral fibrils converge to a series of 'points' atthe upper pole (a/), while additional astral material seems skewed in the oppositedirection at the lower pole.

The lower polar region is shown in greater detail in Fig. 12. Chromosome arms(ch), again identified by surface texture, thickness, and position, can be seen protrudingfrom the fibrous mass. A roughly polygonal structure (cc) is present at the positionexpected for the centriole, adjacent to the fibril convergence point. It appears tohave a central body surrounded by a ring of material which itself has regular sub-structure, with several very thin fibrils leaving the outer edge of the ring at point p.The diameter of the entire structure, including surrounding ring, is approximately0-7 /tm, and its attachment to the rest of the spindle appears rather tenuous. With theSEM such structures have not been readily identifiable at earlier mitotic stages inwhich centriole complexes are most frequently seen in phase contrast. We think itmost likely that they are obscured from view in the SEM by overlying material atthese stages; however, our identification of this polygonal body (Fig. 12, cc) as a'centriole complex' should be regarded as tentative pending further investigation.

For comparison, the same spindle is shown as it appeared under phase contrastafter critical-point drying, before coating (Fig. 11, inset). The 2 highly refractile areas(specimen viewed in air) correspond to the 2 polar regions of greatest density in Fig. 11(SEM).

At telophase (Fig. 13) reconstituting daughter nuclei (n) are seen at the spindle-ends. The spindle now apparently consists entirely of non-chromosomal fibrils,massed together into a compact interzonal bundle measuring about 0-9 /tm in widthat its midpoint, and approximately 13-5 /mi in length. The entire structure seemstwisted, as if one end of the spindle had been rotated with respect to the other. Anindividual fibril (/) can be seen following a route corresponding to such a twist. Onthe surface of the daughter nucleus at the left in Fig. 13, and seen at higher magnifica-tion in Fig. 14, there are a number of ring-like features (r) visible which measureabout 0-25-0-3 /tm in diameter. Structures which might correspond to centriolecomplexes have not been seen at this stage.

Drosophila spindles in phase contrast and SEM 51

DISCUSSION

Properties of D. melanogaster spindles isolated in TPM

Use of an isolation medium based upon tubulin-polymerizing conditions andcontaining protease inhibitor (TAME) and detergent (Triton X-ioo) as described byRebhun et al. (1974) makes possible the preparation of D. melanogaster mitoticspindles suitable for examination with the SEM. Under phase contrast such spindlesare clean; that is, their fibrous structure is readily apparent, as are chromosomes,midbodies, and centriolar complexes. This lack of contamination with other cyto-plasmic material is most likely due not only to the selective stabilization of microtubule-containing structures, but also to the dispersive action of the detergent on membranouscomponents.

The morphology of these spindles after isolation is remarkably faithful to thatreported for blastema-stage spindles in situ (paraffin sections) in the older literature(Huettner, 1933 ; Rabinowitz, 1941). This includes details such as midbodies andtrailing chromosome arms in anaphase. The latter were identified by Rabinowitz(1941) as the long arm of the X chromosome (Fig. 4). Bending of the telophasespindle as seen in isolates can also be found in the older in situ micrographs. Onedifference is noted however: polar structures are lacking in our telophase prepara-tions, as judged by phase contrast. According to Huettner (1933), a pair of centrioles ispresent at each pole in situ at this stage, a relatively long distance away from thereconstituting nuclei. This suggests that they are tenuously connected to the telophasespindle, and probably fall off during isolation.

The morphology of spindles at various stages seemed the same under phase con-trast whether GTP was or was not included in the isolation medium. However, GTPdid seem to stimulate formation of background fibrils in the preparations. The latterwere especially prominent in material from non-mitotic embryos containing inter-phase nuclei, and presumably reflected the availability of a polymerizable tubulin poolduring interphase.

In order to study spindle structure with the SEM, spindles must be isolated freeof debris, and must retain the in situ morphology which characterizes different mitoticstages. These criteria are met by the TPM preparations.

Phase-contrast versus scanning electron microscopy

In general, there is good agreement between phase-contrast and SEM observationsin terms of spindle size and shape, indicating that spindle morphology is little alteredduring the SEM preparative steps. In phase contrast, of course, the material iseffectively transparent so that structures such as chromosomes and centriole complexesare readily visible. Comparison of Figs. 1 and 7 shows that this is not the case for theSEM, as expected; in prophase spindles the chromosomes are hidden beneath thefibrous surface. At later mitotic stages there appear rather smooth-surfaced oblongstructures, thicker than surrounding fibrils, which we believe to be chromosomes. Inthe late anaphase spindle (Fig. 11) in which some of these are visible, it is likely thatothers are concealed within the fibrous polar regions.

52 A. Milsted, W. D. Cohen and N. Lampen

The major fibres seen in metaphase and anaphase spindles under phase contrast arefound to be bundles of smaller fibrils in the SEM. Many of these fibrils are inthe thickness range o-i-o-2/<m, which could accommodate a maximum of about6—25 microtubules. (This estimate excludes the thickness of the metal coating,and ignores possible clear zones or inter-microtubule material.) In some instances,fibrils observed in the SEM are thin enough to be accounted for by single metal-coated microtubules (for example, fibril / and the fibrils radiating from point p inFig. 12). The apparently single interzonal fibre observed in telophase spindlesunder phase contrast (Fig. 5) is similarly found to be constructed of smaller fibrils

(Fig- 13)-The structures seen at spindle poles under phase contrast and referred to as

centriole complexes have a maximum dimension of approximately 0-7 /tm, and thecentral black body of polar regions isolated from cooled embryos is about the samesize. This value seems too great to be accounted for by a simple centriole or centriolepair, even if measurement error is considered. Mahowald (1963) reported that thecentrioles of Drosophila melanogaster in the blastula stages were o-i6/tm in diameterand 0-15-0-175 /tm long, and were never found in fully formed pairs. Fullilove &Jacobson (1971) and Fritz-Niggli & Suda (1972) show centrioles surrounded bysatellites in the forming blastoderm of Drosophila montana and in spermatocytes ofDrosophila melanogaster, respectively. The diameter of these complexes is in therange 0-6—0-8 /.cm, which correlates well with the size of the structures seen at polesunder phase contrast.

In the polar position of some spindles the SEM reveals a structure of roughlypolygonal shape, about 0-7 /un in diameter, with a central body approximately 0-2 /tmin diameter (Fig. 12). This object could be a complex of centriole plus accessorymaterial, possibly satellites, which would serve as the polar microtubule organizingcentre for these spindles (Pickett-Heaps, 1969). While satellites have rarely beenreported surrounding mitotic centrioles (de Harven, 1968), their presence has beenshown around meiotic centrioles of D. melanogaster (Fritz-Niggli & Suda, 1972) andjellyfish (Szollosi, 1964).

One structure seen easily in phase contrast but not yet identified in the SEM is themidbody. While its absence could indicate loss of some midbody material duringSEM preparative steps, a different explanation may hold : the midbody, as seen inphase contrast, could simply be a region of increased density due to overlap of inter-zonal microtubules of opposite polarity and/or inter-microtubule material (Paweletz,1967 ; Mclntosh et al. 1975). Such differences in internal density would not beexpected to appear under the SEM.

An interesting feature of the telophase spindles is the substructure at the surfaceof reconstituting nuclei seen in the SEM (Fig. 14). There appear to be coils or ringsof material, about 0-25-0-3 /tm in diameter. In a study of isolated mouse liver nucleiwith the SEM, Kirschner et al. (1975) found that nuclear pore complexes are resistantto treatment with Triton X-100. However, the features described here are severaltimes larger than typical pore complexes. It is possible that they are exposed chroma-

Drosophila spindles in phase contrast and SEM 53

tin coils, but further work is obviously needed, using a higher resolution SEM onisolated telophase and interphase nuclei.

Events of the mitotic cycle in D. melanogaster embryos

The SEM reveals the progressively more ordered fibrous structure of spindles atdifferent stages of the mitotic cycle. The relatively thin and somewhat tangled fibrilsof prophase assume a more linear arrangement in late prophase and are organized intobundles at anaphase. By telophase, one thick interzonal bundle remains. In metaphaseand anaphase, as seen in phase contrast, there is a fairly small number of major fibresvisible, perhaps 3 or 4. Four midbodies, but not more, have been seen in a number ofanaphase spindles, again suggesting that there may be 4 major fibres involved. SinceDrosophila and other Diptera exhibit the phenomenon of somatic pairing of homo-logues during the mitotic cycle (Metz, 1916), there will be 4 pairs of chromosomes perhalf-spindle in D. melanogaster (zn = 8). The number of major fibres may therefore besignificant, suggesting a relationship between each chromosome pair and a major non-chromosomal fibre. It is noteworthy that the trailing chromosome arms of anaphasespindles often seem associated with one of the interzonal fibres (for example, Fig. 4);this could be fortuitous, however.

In the transition from anaphase to telophase separate interzonal fibres disappear,giving the impression that they associate laterally to form the telophase interzonalfibril bundle. The one large midbody at telophase would then be interpreted as thefusion product of separate smaller anaphase midbodies. The telophase fibril bundleseen in the SEM is about 0-9 /un thick, and this is sufficient to accommodate all of theanaphase interzonal fibrils if they were to associate laterally. Since there is no cyto-kinesis at the blastema stage of development, the presence of midbodies indicatestheir direct involvement in mitosis, possibly as regions of overlap between non-chromosomal microtubules of opposite polarity which have been implicated in asliding spindle elongation mechanism (Pickett-Heaps, McDonald & Tippitt, 1975).

The observed bending of telophase spindles might be significant in relation tospindle mechanics. The SEM reveals an apparent twist of the interzonal fibre bundle,suggesting that the 2 half-spindles rotate with respect to each other.

Use of scanning electron microscopy in analysis of spindle structure

The SEM improves upon both resolution and depth of field obtainable with lightmicroscopy, providing a 3-dimensional view of the entire spindle. Comparable spindlereconstructions from serial thin sections and TEM are difficult to achieve and requireexcessive amounts of labour by comparison. For examination of general structuralorganization of isolates, the SEM might serve as an alternative to high voltage trans-mission electron microscopy as well (Mclntosh et al. 1975).

With respect to small sample preparation methods for the SEM, use of polylysine-coated coverslips (Mazia et al. 1975) permits the following of individual spindlesunder phase contrast through all of the processing steps. They can be photographedafter critical point drying for subsequent identification in the SEM using spindleorientation and adjacent landmarks. Polylysine-coated plastic coverslips have proved

54 A. Milsted, W. D. Cohen and N. Lampen

more convenient for our purposes than either polylysine- or Formvar-coated glass(Lung, 1974). While adequate for the work thus far, significant losses of material havebeen encountered ; perhaps better retention of material might be achieved by covalentlinking to the substratum (Vial & Porter, 1975).

To our knowledge, this is the first study of spindle structure in which the SEM isutilized. Thus we have not had the advantage of comparison with the data of others.However, based on our results, we believe that the SEM will become an increasinglypowerful tool for such work as improvements are made in the areas of instrumentresolving power, specimen retention, and reduction or elimination of the metalspecimen coating.

We wish to express our appreciation to Dr Etienne de Harven for use of the CambridgeScanning Electron Microscope and preparative facilities, for his interest, and for critical readingof the manuscript. This work was supported in part by Faculty Research Award no. 1220 of theResearch Foundation of The City University of New York, and by grant no. GB-25578 of theNational Science Foundation to Dr W. D. Cohen. Submitted in partial fulfilment of therequirements for the Doctor of Philosophy degree at The City University of New York (A. M.).

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{Received 27 January—Revised 3 June 1976)