cytochemical adenosinetriphosphatase in ...j. cell set. 3, 423-436 (1968 42) 3 printed in great...

J. Cell Set. 3, 423-436 (1968) 423

Printed in Great Britain

CYTOCHEMICAL ADENOSINETRIPHOSPHATASE

IN PLANT ROOT MERISTEM

NORMA SHIFRIN AND L. LEVINELaboratories of Cell and Molecular Biology, Department of Biology, Wayne StateUniversity, Detroit, Michigan 48202

SUMMARY

Root tip meristems were stained to demonstrate ATPase activity by two different methods,with general agreement in localization but not specificity, and with emphasis on mitotic cells.

In interphase, ATPase was localized in nucleoli and nuclear membranes, with lesser activityin the nuclear substance. In prophase, chromosomes were outlined by ATPase stain whichgradually declined in intensity at prometaphase, becoming least evident in metaphase. Stainingactivity increased again in anaphase, and remained high in telophase. In prometaphase, ana-phase and late anaphase-early telophase, the ATPase was concentrated in a fibril which appearedto coil around the chromosomes. The ATPase fibril was thinnest at metaphase and shorter andthicker at telophase. In addition, granules farmed in association with the coils of the fibril inlate anaphase and early telophase. Later on, these granules may have fused and contributed tonucleolar reformation.

The ATPase never localized in the chromosomal fibre nor in any other region of the spindle.RNA generally localized like ATPase, but ATPase loci were unchanged after ribonuclease(RNase) treatment.

Because of certain similarities between ATPase and argentaffin localization, some relationshipbetween the nucleolus and ATPase is suggested. A mechanochemical transducing role is postu-lated for the ATPase, because cytochemical properties were like those of ATPase in the A-bandof myofibrillae, and because other changes in it could be correlated with chromosome movement.

INTRODUCTION

Adenosinetriphosphatase (ATPase) activity appears to be a consistent property ofmany mechanochemical systems. It cannot be dissociated from the H-meromyosinfraction obtained in appropriate extracts of muscle (Mihalyi & Szent-Gyorgyi, 1953).ATPase is demonstrable, as well, in comparable extracts of non-muscular cells: sarco-matous fibroblasts (Hoffman-Berling, 1956), human thrombocytes (Bettex-Gallard &Liischer, 1961), slime mould plasmodia (Nakajima, 1964), and isolated cilia (Gibbons& Rowe, 1965; Culbertson, 1966).

In striated myofibrillae, ATPase may be cytochemically localized in the A-band(Padykula & Gauthier, 1963; Gauthier & Padykula, 1965) where myosin is concen-trated (Huxley & Hanson, 1957; Hanson & Huxley, 1957). Cytochemical localizationof ATPase in sperm flagellae (Nelson, 1958; Nagano, 1965), certain fibrils of myxo-mycete plasmodia (Wohlfarth-Bottermann, 1964) and the myonemes of Vorticellans(Levine, i960) is also reported.

The mitotic apparatus (MA) appears comparable to mechanochemical systems inthis aspect. Adenosinetriphosphate (ATP) produces anaphase elongation in glycerated

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424 N. Shifrin and L. Levine

fibroblasts and contributes to the completion of a cytokinetic furrow (Hoffman-Berling, 1954, 1964). ATPase is present in the isolated MA of sea-urchin eggs (Mazia,Chaffee & Iverson, 1961; Miki, 1963). Cytochemically, it is more concentrated in themetaphase and anaphase MA than in the rest of the cytoplasm (Miki, 1963). Finerlocalization of ATPase within the MA of HeLa and Sarcoma 180 tissue cultured cellsis also reported (Hartmann, 1964), in the metaphase chromosomal fibre regions andboth anaphase and telophase interzonal regions.

This paper deals with the cytochemical demonstration of ATPase in plant meri-stems. Its purpose is to compare localizations of ATPase in the MA of cells differentin origin from those previously worked with, to test the general nature of its distri-bution. The results of this investigation indicate that the ATPase is closely associatedwith chromosomes and is not present in chromosomal or continuous fibres.

MATERIALS AND METHODS

Album cepa root tips were obtained from bulbs set in aerated tap water at roomtemperature for 3 or 4 days. Tips, 4 mm long, were cut from roots, 1 to 2 cm long,and immediately dropped into fixative.

Fixation

Pilot experiments showed that fixatives used for sea-urchin eggs (Miki, 1963) andtissue cultured cells (Hartmann, 1964), 80 % ethanol and calcium-formol respectively,did not preserve ATPase in plant tissues. However, glutaraldehyde- and hydroxy-adipaldehyde-containing fixatives, used for the subcellular localization of ATPase inother cells (Sabatini, Bensch & Barrnett, 1963), could be used, if isotonic (Gordon,Miller & Bensch, 1963). Both glutaraldehyde and hydroxyadipaldehyde were adjustedto approximately isosmotic concentrations (that is, equivalent to 190 m-osmoles KCl)with the aid of a one-drop cryoscope (Levine & Musallam, 1964). The compositionsof the isosmotic fixatives used were as follows: 1-55 % hydroxyadipaldehyde, 0-55Msucrose, CV0125M cacodylate buffer at pH 7-5 (HAA); and 1-3 % glutaraldehyde,O-OO2M CaCl2, 0-025M cacodylate buffer at pH 7-2 (Gl). The best fixation obtainedwith Allium tissue was in a mixture of one part GL to nine parts HAA (GL:HAA).All tissues were fixed for 5-6 h at o °C. They were subsequently washed three timesin cold isotonic buffer (5-3% sucrose in 0-025 M cacodylate buffer, pH 7-4). Thefixed tissue was stored in isosmotic buffer at 4 °C until used (1 day to 2 months).Since the tissues did not seem to show any appreciable osmotic damage, the fixative-sucrose mixtures used may have been isotonic as well as isosmotic. Fixed tissue wassectioned in a cryostat (International Harris, Model CTD) between — 150 and — 20 °C.Ten longitudinal or 20 cross-sections were then mounted on albuminized coverslips,thawed by finger warmth, and allowed to dry at room temperature for 30-40 minbefore immersion in the assay medium.

Adenosinetriphosphatase in root meristem 425

A TPase staining medium

Two methods were used to demonstrate ATPase activity:(1) Wachstein-Meisel (Wachstein & Meisel, 1957) method (WM). Incubation

medium: 12-5 mg ATP (8-3 x IO~4M), 10 ml o-2M TRIS-maleate buffer, 2-5 ml 2 %Pb (NO3)2, 1-5 ml O-IM MgSO4, n-o ml H2O. The pH was adjusted to 5-2, 5-6, 6-o,6-4, 6-8, 7-2 and 7-6 with o-i N HC1, and the solution filtered. Sections were incubated5-120 min at room temperature, washed in three changes of water, and immersed in a10 % dilution of (NH4)2S (Baker Analysed Reagent, assayed at 23-5 % concentration) for30 sec. Following this, they were washed, and the coverslips were mounted in gly-cerine and ringed with colourless nail polish. Optimum staining occurred at pH 6-4.For this reason, when other substrates were substituted for ATP, the medium wasadjusted to pH 6-4. The substrates were: adenylic acid (AMP), adenosinediphosphate(ADP), inosinetriphosphate (ITP), beta-glycerophosphate (GP), and fructose-1,6-diphosphate (FDP) at 8-3 x io~4M.

(2) Padykula-Herman (Padykula & Herman, 1955 a, b) method (PH). Incubationmedium: 75-6 mg ATP (5 x IO~3M), 10-98 mg cysteine-HCl, 6-25 ml O-IM veronalbuffer pH 9-4, 5 ml o-i M CaCl2, H2O to final volume of 25 ml. Substrate pH was thenchecked and readjusted as necessary. Incubation time was varied between 5 and 120min at room temperature. Following incubation, sections were washed in controlmedium saturated with (Ca)g(PO4)2 by the addition of O-IM NaH2PO4 for 3 min,2 % CoCl2 for 3 min, and three changes of water. Both the CoCl2 and final washwater were brought to pH 8-o by the addition of veronal buffer. Sections then wereimmersed in the 10 % dilution of (NH4)2S (as prepared in WM method) for 30 sec,washed in three changes of water, and dehydrated in 80 % (1 min), 95 % (2 min) and100% (3 min) ethanol. The coverslips were mounted in Permount after two i-minchanges of toluene. Other substrates used were: 5 x io~3MADPand ITP; i-2x IO~2M

AMP, GP, and FDP.Sulphydryl dependence. GL:HAA-fixed sections were incubated in cysteine-free

PH substrate media containing 0-005 M .p-hydroxymercuribenzoate (POHMB) for1 h. (The POHMB was obtained from the Sigma Chemical Co.) Parallel sectionswere also incubated in cysteine-free PH medium without POHMB.

Controls. Parallel sections were routinely incubated in both WM and PH mediawithout substrate. In addition, some sections were steam-treated for 30 min beforeincubation in substrate media. Glass distilled, deionized water was U9ed in all pro-cedures, unless otherwise specified.

RNA and DNA distribution

Both RNA and DNA were visualized in GL: HAA-fixed Allium cryostat sectionsby methyl green-pyronin stain (Kurnick, 1955). Specificity was tested by stainingafter treatment with DNase and RNase solutions, or their appropriate controls (bothof the enzymes were obtained from the Sigma Chemical Co.); 0-02 % DNase in thepresence of 0-003 M MgSO4 was used after adjustment to pH 6-o with O-IN NaOH;RNase was employed as a o-oi % solution at pH 6'5. Control solutions were identical

426 N. Sfrifrin and L. Levine

in each case, except that they did not contain the enzyme. Some sections were sub-jected to the PH assay after being digested with RNase.

We used the usual criteria of chromosomal position (checked by phase contrastwhen necessary) to recognize metaphase, anaphase, and telophase. Seriation withinprophase was judged by nuclear size, and size and shape of the nucleoli (relativelylarge and smooth in outline during early, and proportionately smaller and stellatein late prophase). Diagnostic features for prometaphase were: absence of nuclearmembrane with chromosomes not yet aligned in equatorial plane. Progression throughtelophase was judged by relative increase in condensation of chromatids, presence ofnucleoli, growth of cell plate towards cell walls, and presence of nuclear membrane.Photographs were taken through a Zeiss GFL microscope with a Zeiss Planapox 100 oil 1*3 n.a. objective and Apl 1-4 n.a. condenser.

RESULTS

ATPase: PH and WM

A specific locus was considered to contain active enzyme when a definite depositappeared after incubation in substrate-containing media, but did not appear, or wasonly slightly evident after incubation in substrate-free media. Such deposits weredetectable after a minimum of 5 min; maximum deposition occurred after 1 h incu-bation. The texture and colour of deposits differed in WM and PH. Deposition wasfiner and black with PH; it was relatively coarse and dark brown to black after WM.WM gave positive results with GL but not with HAA fixation. On the other hand,PH had to be used after HAA or GL:HAA since GL gave positive controls.But, GL:HAA was used routinely for PH since it produced superior cytologicalpreservation.

PH

During interphase, there was slight activity on the nuclear membrane. Nucleolihad much darker deposits (Fig. 1) with an occasionally lighter interior. In earlyprophase nuclei, deposition occurred in fine granules which seemed to be organizedin a net, speckled in places with nodules. As prophase progressed into prometaphase,the ATPase outlined larger unreactive spaces, nodules were still evident, and thestellate nucleolus appeared, at some points, to be continuous with the activity out-lining clear spaces (Fig. 2). These clear spaces were the chromosomes (as shown byMGP), negative for ATPase. Somewhat later on in prometaphase and early metaphase, alighter deposition surrounded the chromosomes, which in longitudinal sections seemeddisposed in very fine granules or as a continuous line (Fig. 3). In addition, several fineATPase active fibrils crossed some of the chromosomes (Fig. 4).

Chromosomal ATPase continued to decline in metaphase. In fact, most metaphasecells were difficult to distinguish without phase contrast, with few exceptions. Theonly activity in these exceptional cells outlined the chromosomes (Fig. 5).

In early anaphase (Fig. 6), chromosomal ATPase seemed to produce denser depositsthan in metaphase. The chromosomal ATPase was relatively more intense in later


than in earlier anaphase, giving the chromosomes a banded appearance with a dis-tinctly granular outline, depending on their orientation in the plane of focus (Fig. 7).

Perfectly flat longitudinal sections of chromosomes were rare, and careful focusingalong the length of the obliquely oriented chromosomes was necessary in order toattempt a three-dimensional reconstruction of the ATPase-active structures whichsurrounded them. Many chromosomes were studied, and careful focusing revealedthat granules on one side of the chromosomes were joined with granules on the otherside by fine interconnecting fibrils. The granule image could be followed along thefibril, blending into it as if the granules were end-on views of the fibrils. Thesegranules, therefore, are only apparent. They represent a section through the fibrilwhere it curves around the chromosomes. For this reason, we think that ATPase iscontained within a fibril which encircles the chromosomes, most commonly as ahelix (Figs. 7, 8). In some chromosomes, the helix was only evident at the distal end,but then meandered towards the centromeric end along its sides, looping over onlyoccasionally. This ATPase-active fibril very closely resembles the nucleolonema inmitotic cells (Estable & Sotelo, 1954).

In late anaphase and/or early telophase (Figs. 9, 10), activity outlining the chromo-somes was intense and appeared almost as a continuous band. Within this, it waspossible to resolve the closely spaced circular cross-sections of the helical fibril.Occasionally at least one granule per chromatid mass could not be reconstructed as afibril cross-section. This type of granule formed with distinct relation to one of thecoils (often appearing continuous with it) of one chromosome. Frequently, anothergranule could be found, symmetrically placed in the other chromatid mass, but thisdid not always appear to be of equal size. Later on, as chromosomes coalesced witheach other, ATPase was confined to small nucleoli and to relatively larger granuleswhich were often aligned in rows (Figs. n-13). Some of the smaller nucleoli werecontinuous with the activity that seemed to outline chromosomes (Fig. 11). As thecell wall appeared, these ATPase granules were fewer in number; they were neitherspherical (Fig. 12) nor of regular size (Fig. 13). Their large size, their irregular shapes,and the increased space between them gave the impression of fusion with each other.These telophase granules were different in character from those of prometaphase orearly and mid anaphase. They were larger and denser and could not be interpretedas fibre cross-sections. By interphase, the granules were no longer present.

The cytoplasm of cells from late prophase to late anaphase often appeared darkergrey than the cytoplasm of cells in other stages (Fig. 5), but in no case was the ATPaselocalized in regions of the spindle (e.g. chromosomal fibres).

SH-Dependence and specificity

Sections treated with POHMB were inhibited in every case, but not always to thecontrol (no substrate) level of staining (Fig. 14). This figure also shows the typicallevel of control nucleolar staining. These nucleoli, however, were considerably lighterthan those incubated with ATP. In addition, steam pre-treatment almost abolishedtheir staining after incubation in control media. No activity above control level wasdetectable when other substrates were substituted for ATP in the incubation media


(Table 1). Therefore, PH was as specific with root tips as with the animal tissues forwhich it was originally developed (Padykula & Herman, 19556).

WM Method

Enzyme-active sites, identical to those already described under PH, were found withWM (Figs. 15-18). In addition, cytoplasmic granules were distributed during inter-phase and prophase around the nucleus in meristematic cells. In some cells, however,there was a greater granular accumulation at opposite ends of the cell (Figs. 15, 16).During later stages of mitosis, granular staining either outlined the mitotic spindle orwas predominantly polar. However, most cells showed polar distribution of activity(Fig. 17). Occasionally, granules were visible in the interzonal area in late anaphase(Fig. 18). Nucleoli stained moderately brown. Controls were completely negativeexcept for light deposition in nucleoli.

Table 1. Substrates demonstrating phosphatase activity in Allium cepa withPadykula-Herman and Wachstein-Meisel techniques

Substrate PH WM

ATP + + + +ITP - + +ADP - + +AMP - +GP - + +FDP

— = no stain; + = light stain; + + = moderate stain

Specificity

Splitting of inorganic phosphate from substrates other than ATP could be demon-strated. The results are summarized in Table 1. ITP, ADP, and GP produced inten-sities and localizations identical to ATP. Staining also resulted, to a lesser degree, withAMP. There was no staining with FDP. Therefore, WM was less specific than PH.

MGP

The characteristic pyroninophilia of RNA was seen in nucleoli and cytoplasm ofinterphase cells. Chromatin stained blue (Moss, 1966). In mitotic cells the chromo-somes, taking on a purple hue, were embedded in an RNA matrix which occupied thespindle area. They tended to be more purple in metaphase and anaphase than inprophase and prometaphase. Chromosome arms protruding from the spindle area, orin cross-section, had a reddish outline. In telophase, groups of coalescing chromo-somes contained small RNA granules aligned in rows (as in Fig. 22), as well as one ormore larger ones. Their alignment in early telophase tended to resemble that of theATPase telophase granules, but the latter seemed more numerous and prominent.

RNase digestion before MGP removed pyronin staining, but the results werepatchy. Some areas of any one section had a residual faint pink tint, interspersed at


random with areas having no such residual stain. In cells with no detectable remainingstain the chromosomes were blue. This is in contrast to the undigested MGP-stainedchromosomes, which tended to be purple from metaphase through early telophase.We believe that the origin of the purple may have resulted from a red cast on the bluechromosomes, indicating that these chromosomes were coated with RNA. RNase hadeffects other than upon the pyroninophilia; it produced some uncoiling of telophasechromosomes.

When DNase treatment preceded MGP, the chromosomes themselves were un-stained but were outlined by a thin pyronin-pink line, from prophase through earlytelophase (Figs. 19-22). In addition, fine pink fibrils looped around some chromo-somes (Figs. 20, 21). The fibril was also indicated in MGP alone, but contrast of thedelicate fibril against the darkly staining chromosomes was too weak to permit un-qualified identification. In late telophase, RNA was concentrated in granules dispersedwithin the reforming nucleus (Fig. 22), as in MGP alone. The 'wispy' RNA-containing lines interpreted by Swift (1959) as spindle remnant were also seen.DNase caused the chromosomes to swell. With the possible exception of telophasegranules, RNA seemed to localize on the chromosomes in the same way as the ATPase.

After incubation in RNase and control media (without RNase), chromatin oc-casionally displayed a loosely aggregated greyish stain in the PH method (with andwithout substrate). Since this staining was dependent neither upon previous treatmentwith RNase nor upon the presence of ATP in the medium, it is considered to be theresult of non-enzymic processes.

DISCUSSION

Loci for ATPase, established with PH, were reproduced by the less-specific WMmethod. WM also revealed additional activity in cytoplasmic granules. These granuleshad a polar distribution during metaphase and anaphase; but other cells also had thispolarized distribution in interphase and prophase. It may be that the distributionduring later stages of mitosis resulted from the herding of cytoplasmic inclusions tothe ends of the cell by the growing MA which eventually occupies almost the entirevolume of the cell. The distribution during interphase and prophase may be a relicof the previous anaphase. While no definitive identification of these granules wasmade, their ability to stain near neutrality in the absence of exogenous SH indicatesthat they may be mitochondria (Padykula & Gauthier, 1963).

Comparative localizations of ATPase in plant and animal MA

The localization of ATPase in mitotic meristematic cells was not within the chromo-somal or continuous fibre regions of the spindle, and therefore differed from thelocalization in some animal cells (Miki, 1963; Hartmann, 1964). Miki's biochemicaldata confirmed those of Mazia et al. (1963), and, in addition, provided other propertiesof the ATPase. Stephens (1967), however, did not find ATPase in either hexyleneglycol spindle isolates (Kane, 1967) or the major highly purified soluble 22 s com-ponent protein. There is a possibility that the enzymic activity demonstrated by cold


ethanol-sonication (Miki, 1963) and dithiodiglycol (Mazia et al. 1961) methods mayhave resulted from a non-fibrou9 matrix component carried along with the isolates.Yet it cannot be excluded that the hexylene glycol preparatory procedures may haveadversely affected the ATPase. In any case, Miki's observations showed ATPasemore concentrated in the spindle area than in the surrounding cytoplasm. The use ofwhole mounts, however, coupled to the large numbers of small chromosomes presentin sea-urchin eggs, made definitive cytological localization impossible. These data,therefore, could not show precisely where in the spindle or chromosomes ATPasemay be localized. Hartmann (1964), however, was more concerned with cytologicalobservations, per se. He found ATPase generally within the metaphase spindle ofHeLa and Sarcoma 180 cells. Frequently, but not always, a small intense area ofactivity was found at one or both HeLa spindle poles. Polar views of metaphaseSarcoma 180 cells showed granular localization, apparently in the kinetochore region.The large numbers of small chromosomes in relatively small cells made it impossibleto determine the exact relationship of the activity to the chromosomes, and the polarviews did not permit unequivocal establishment of stage (they could have been ana-phases). ATPase of HeLa metaphase spindles was too intense to differentiate ordersof activity within the MA. In telophase, ATPase localized in the interzonals.

In summary, these observations differ from those made with meristematic cells asfollows: meristematic cells showed no activity in the spindle area, while sea-urchineggs and sarcomatous cells did have this activity; and meristematic ATPase was minimalwhile sarcomatous ATPase was high during metaphase. Yet there are certain simi-larities. Chromosomes did not stain in either case, both meristematic and mammaliansarcomatous ATPase was SH-dependent, and, while the SH-dependence of sea-urchinATPase was not tested directly, the distribution of its ATPase followed the distribu-tion of SH found by Kawamura & Dan (1958). In addition, there is the possibilityfor chromosome-associated ATPase in Sarcoma 180 cells. In one figure (fig. 6, Hart-mann, 1964), fine lines of activity seemed to extend between chromosomes. Finally,both late anaphase meristematic cells (with WM only) and Sarcoma 180 cells haveactivity in the interzonal regions.

The differences in localizations are difficult to reconcile, and perhaps final analysesshould be reserved until all tissues concerned may be fixed by identical methods. Itmust be borne in mind, however, that the meristematic and typical animal spindlesdo differ, since, as is well known, animal spindles have a centriole at each pole. Thedifferences in ATPase localization, therefore, may be real and may reflect this dif-ference in organization of an enzyme of apparently similar cytochemical properties.Unqualified support of this hypothesis must await further experiments, such as theone suggested. Attempts to fix root tips by the methods of both Miki and Hartmannyielded unsatisfactory results. We also considered the possibility that the fixation didnot adequately preserve spindle structure, and that this was the reason for its lack ofstaining. But tissues fixed in GL: HAA and stained by MGP showed that the RNA-rich spindle was present. Furthermore, fibrous elements were visible in the half-spindles of cells post-fixed and then stained with fast green. Chromosome-associatedATPase may have been activated by glutaraldehyde as reported by Dejong, Olson &


Jansen (1967) for nuclear acid phosphatase of cultured tobacco cells. This is con-sidered unlikely, since we would expect such activation in metaphase, where activityis practically absent, to be comparable with other phases.

ATPase and the nucleolus

The relatively high levels of nucleolar control staining obtained was also noted byothers (NovikofT, Essner, Goldfischer & Heuss, 1962). Control nucleolar stainingmay result, in part, from its RNA content, which could provide a potential source ofpolyphosphate, or from its contained calcium (Steffenson, 1961). Tandler (1954)showed that nucleoli bound cobalt, although at lower pH's than we used. She con-sidered this cobalt-positive substance to be a distinct nucleolar fraction. Our observa-tions on steamed sections, which markedly reduced control deposition, suggest addi-tional involvement of endogenous substrate.

Danielli (1953) considered alkaline phosphatase as an almost universal property ofnucleoli. Coleman (1965) demonstrated, by biochemical methods, three nuclearATPases. These showed, by electron-microscopical cytochemical methods, denserdeposition over nucleoli than over chromatin fibrils. One of these was a calcium-activated ATPase with pH optimum of 8-4.

There are striking parallels between ATPase localization and the distribution ofnucleolar specific silver staining (Das, 1962; Estable & Sotelo, 1954; Tandler, 1954,1959). According to Das (1962) there was no silver reduction by the condensed chro-mosomes from prophase through anaphase; and it was not until early telophase thatsilver-stained granules appeared around all the chromosomes. He noted an increaseof cytoplasmic silver staining at about the time of nucleolar dissolution, and a corres-ponding decrease when telophase granules reformed which led him to suggest apossible shift of silver reactive substance from nucleolus to cytoplasm and back againinto the nucleolus at the time of its reformation. Tandler (1954, 1959) did not findcytoplasmic silver staining, and outside of this, her work was largely confirmed byDas, but for one additional observation. She described (1959) a very thin layer ofsilver-staining substance with a ' chromatin-like' distribution in late anaphase-earlytelophase. In more advanced stages, small scattered drops formed, and these werethought to fuse into several masses of prenucleolar bodies. Neither Das nor Tandlerconsidered the silver-staining substance to be RNA. Furthermore, they could notfind any evidence of chromosome-associated nucleolar substance that persisted throughall the mitotic stages. Estable & Sotelo (1954), however, did describe such a fraction,the nucleolonema. According to these authors, the nucleolonema, present in interphaseas a complicated glomerulus, becomes unravelled on to the chromosomes for transportbetween generations as a fibre in longitudinal or helical association with them. It isthinner at metaphase, and becomes shorter and thicker at telophase, when, along itslength, spherules form which eventually aggregate and fuse to reform the interphasenucleolus.

Our observations on ATPase localization were practically identical to those madefor the silver staining by Das and Tandler during late anaphase and telophase, andby Estable & Sotelo during all the mitotic stages. Although RNA localized in the


same way as ATPase, ATPase localization did not appear to be altered by RNasedigestion. The cytoplasm of cells in late prophase through telophase showed moreactivity than cells in interphase. Besides being in interphase nucleoli, ATPase out-lined prophase chromosomes, in apparent continuity with nucleoli. Our descriptionsfor the ATPase fibril on chromosomes are almost identical to those of Estable &Sotelo for the mitotic nucleolonema; both had practically the same morphology atsimilar mitotic stages, both were least evident at metaphase, and both were shorterand thicker at telophase. The regularly spaced anaphase ATPase granules seemed toform in close geometrical relation to the coils of the chromosome-associated ATPasefibril, which gave them their linear array. They resembled, in geometry, time ofappearance, and RNA content, the chromosomal RNA described by Swift (1959).Their regularity argues against their being spindle remnant (another distinct RNAfraction described by Swift) since this would be expected to be incorporated in a morerandom manner. Besides, these bodies contained ATPase, and the spindle did notcontain ATPase. There are a number of possible origins for these granules. TheATPase fibril changed configuration and sometimes appeared in apparent continuitywith the granules; cytoplasmic ATPase decreased; and nuclei may be beginningcapabilities for accumulation of chromosomal and nucleolar total protein and nucleolarRNA (as judged for another species, Viciafaba, by Woodward, Rasch & Swift, 1961).Our data do not permit discrimination between these or some other origin of thegranules. Later in telophase, the ATPase granules decreased in number as nucleolireformed. Their shapes suggested that they fused to form larger bodies. Lafontaine(1958) and Lafontaine & Chouinard (1963), working with plant root meristem, couldrecognize nucleolar substance by its characteristic fine structure. Small bodies withthese characteristics were first noted in late anaphase, and these seemed to fuse duringtelophase giving rise to mature interphase nucleoli (however, cf. Swift, 1959). There-fore, the small bodies with characteristic nucleolar fine structure were called pre-nucleoli. In an electron-microscope study of grasshopper neuroblast cells, Stevens(1965) suggested that the interphase nucleoli resulted from the fusion of pre-nucleolarbodies also identified by characteristic fine structure. Tandler (1959) interpreted telo-phasic droplets as prenucleoli, because they gave evidence of fusion and containedspecific argentaffinic nucleolar substance. It would appear, therefore, that the regularlyaligned late anaphase ATPase granules could also be called prenucleoli, because theyappeared to fuse to form larger bodies in late telophase, resembling interphasenucleoli by their ATPase content.

Our data therefore suggest that the ATPase is localized in structures resemblingproposed forms of nucleolar substance during mitosis. They also indicate that ATPaseloci exhibit structural continuity between generations.

Possible role(s) of A TPase in mitosis

That the nucleolus may have intimate functional relationships to the mitotic pro-cess has been shown by Gaulden & Perry (1958) in grasshopper neuroblasts. Intenseu.v.-microbeaming of the nucleolus (from late telophase to the middle of mid-


prophase) permanently stopped mitosis. These authors also reviewed pertinentliterature on plants. However, von Borstel & Rickenmeyer (1958) found that mutantDrosophila melanogaster embryos, lacking nucleolar organizer chromosomes, werecapable of 10-12 zygotic mitoses. They suggested that organized nucleoli may beunnecessary in these very rapid divisions in which nucleoli do not normally develop.It would be of interest to obtain silver-staining data for these mitoses.

In any case, Kusanagi (1964) traced ^HJcytidine pulse-labelled cells in Luzulapurpurea root meristem through mitosis. Some of his results led him to believe thatnucleolar RNA moved on to the chromosomes in late prophase and prometaphase.When similar labelling followed irradiation (Kusanagi, 1966), almost all radiation-induced akinetic fragments were unlabelled while all the kinetic fragments werelabelled. Since one source of label was the nucleolus, Kusanagi hypothesized thatnucleolar substance may impart kinetic ability to the chromosomes.

We believe that the chromosome-associated ATPase, localized on meristematicchromosomes in nucleolar-related structures, has some bearing on their kinetic ability.Most motile systems investigated (see Introduction and Weber, 1958, for review)have an associated ATPase, and ATP induces aspects of mitosis in glycerated cellmodels (Hoffman-Berling, 1956, 1964). In particular, chromosome-associated ATPaseand ATPase in sarcomatous tissue-cultured cells showed properties in common withthe ATPase localized in the A-band of myofibrillae (Padykula & Gauthier, 1963;Gauthier & Padykula, 1965). Both were active at pH 9-4 in the presence of exogenouscysteine-SH, and both were inhibited, although not to the same degree, by PCMB.In meristematic cells, the activity was specific for ATP, and did not hydrolyse othersubstrates. In addition, changes in activity or conformity of active loci occurred whenchromosome motion would change. Thus, in metaphase, when chromosomes wouldhardly be moving, the chromosome-associated ATPase was least active, and/or activeloci were thinnest. This suggests a diminution of mechanochemical transduction atmetaphase. During anaphase, chromosome-associated ATPase was most active and/oractive loci were thicker and shorter (the coils were closer together).

If ATPase reveals sites of mechanochemical transduction, then these sites wouldhave to be localized on the chromosomes, and not in chromosomal fibres. Accordingly,various theories of chromosome motion, implicating chromosomal fibres as primaryforce-producing structures (see Schrader, 1953; Mazia, 1961; Roth, 1964 for reviews;and Roth, Wilson & Chakraborty 1966) may have to take this into consideration. Forer(1965,1966) already discussed the need for re-evaluation of the evidences upon whichtraction fibre hypotheses are based, since the results of his u.v.-microbeaming ex-periments suggested, among other things, that birefringence may be separated fromtraction in the chromosomal fibre. Yet some of his data led him to consider chromo-somal fibres as the sites of force production (or transmission).

We do not necessarily exclude other roles for the ATPase, such as a chemo-syntheticone for RNA-protein, or some other structural role as in the orientation of spindlesubunits in endothermic equilibria (Inoue, 1964). Some part or all of the ATPasemay be engaged in these or other unknown roles, but we tend to favour the mechano-chemical one at present, because of the cytochemical parallels that may be drawn with


myofibrillae, and the changes in conformity of chromosome-associated ATPase withchromosome motion.

This paper is Publication Number 190 of the Department of Biology, Wayne State University.We wish to express our appreciation to Dr James E. Perley for his critical review of this

manuscript, and to acknowledge pre- and post-doctoral fellowships (U.S. Public HealthService F1-GM-14655 and F2-GM-14655) granted to one of us (N.S.).

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(Received 16 October 1967—Revised 24 February 1968)

Allium cepa. Figs. 1-7, 11-22, magnification X1700; Figs. 8-10, magnificationx 2200. Figs. 1-14, ATPase activity by Padykula-Herman technique.

Fig. 1. Cells in interphase (a) and early prophase (b). ATPase activity is localized inthe nucleolus and nuclear membrane. Within the nucleus of early prophase cells, theactivity appears in a granular net with occasional nodules (arrows).Fig. 2. Late prophase. ATPase-active net outlines the chromosomes and appears to becontinuous with a stellate nucleolus. Arrows point to nodules.Fig. 3. Prometaphase or early metaphase. ATPase activity appears either as a con-tinuous outline around the chromosomes or as fine granules (arrows). Small teardropnucleolus shows apparent continuity with activity around chromosomes.Fig. 4. Prometaphase or early metaphase. Chromosomes outlined by ATPase activity.Activity is also evident in granules along chromosomes (arrow) and in fibrils acrossthe chromosomes (double arrow). Shadows in chromosomal region are produced bychromosomes out of the plane of focus.Fig. s. Metaphase. The light ATPase activity around the chromosomes is either con-tinuous or granular (arrow). Note light cytoplasmic activity in interphase cells (doublearrow) as compared with activity in metaphase cell.Fig. 6. Early anaphase. Heavy ATPase activity is around the chromosomes in a con-tinuous outline (double arrow), in granules (arrow), and in fibrils crossing the chromo-somes (/).Fig. 7. Mid-anaphase. Intense ATPase activity in fibrils gives chromosomes bandedappearance (double arrow). Note that the apparent granules are continuous with thefibrils crossing the chromosomes (arrow). These granules are interpreted as cross-sections of the fibrils.

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N. SHIFRIN AND L. LEVINE (Facing p. 436)

Fig. 8. Same cell as in Fig. 7, in different level of focus. Note helical nature of ATPasefibrils (arrow).Fig. 9. Late anaphase-early telophase. Activity is distributed as in earlier anaphase.On some chromosome arms, cross-sections of fibrils appear closer together (arrow).Granule may be seen forming on coil of one chromosome (g).Fig. 10. Late anaphase-early telophase. Cross-sections of ATPase helix are closeenough to almost touch each other (arrow). Granules may be seen forming on coils ofchromosomes (g).Fig. 11. Early telophase. Larger granules are present between chromosomes (arrows).Reforming nucleolar bodies (n) seem to be continuous with the interchromosomalstaining.Fig. 12. Mid-telophase. Nucleolar bodies are larger. The larger size, increase in spacing,and the irregularities in shape of some granules give the impression of fusion (arrows).Fig. 13. Late telophase. Activity is present in the nucleoli and in the fewer, morewidely spaced, different-sized granules (arrows) in reconstituting nuclei.Fig. 14. Late telophase. Control section. There is light staining of nucleoli and gran-ules within nuclei.

Figs. 15—18. ATPase activity by Wachstein—Meisel technique.Fig. 15. Interphase. Heavy deposition of stain localizes in the nucleolus and in the

cytoplasm at the polar regions of the cell (g). There is some light granular staining innucleus.

Fig. 16. Prophase. ATPase activity appears in nucleolus, nuclear membrane, and incytoplasmic granules (g) in polar regions. In the nucleus, a granular net with occasionalnodules (arrows) can be seen.


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Fig. 17. Metaphase. ATPase-active granules accumulate in a predominantly polarposition. Faint chromosome-associated stain is visible.

Fig. 18. Anaphase. The ATPase activity appears in very fine fibrils which crosssome chromosomes (arrow), and between chromosomes as granule-like depositions(double arrow) which are presumably cross-sections of these fibrils. Interzonal areacontains ATPase activity.

Figs. 19-22. Allum cepa sections subjected to methyl gTeen-pyronin staining aftertreatment with DNase for 1 h. Only red stain of pyronin is observable.

Fig. 19. Prophase. Pyronin staining appears in nucleolus, in net outlining chromo-somes (arrow), and in cytoplasm.

Fig. 20. Prometaphase. Pyronin staining occurs around chromosomes and acrossthem in fine fibrils (arrows).

Fig. 21. Anaphase. There is diffuse pyronin staining in entire spindle area withinwhich chromosomes are outlined. Arrows point to fibrils crossing the chromosomes.

Fig. 22. Telophase. Pyronin staining occurs in reforming nucleoli (n), and in dis-continuous rows of granular texture within the reconstituting nuclei (arrows).

N. SHIFRIN AND L. LEVINE

cytochemical adenosinetriphosphatase in ...j. cell set. 3, 423-436 (1968 42) 3 printed in great...

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