THE DETERGENT-RESISTANT CYTOSKELETON OF ...Fibrillar proteins have long bee by electron knownn microscopy, to pervad, e cytoplasm but the advent of detergent-extraction techniques

J. Cell Set. 56, 3I9-33S (198a) 319Printed in Great Britain © Company of Biologists Limited 1982

THE DETERGENT-RESISTANT CYTOSKELETON

OF HIGHER PLANT PROTOPLASTS CONTAINS

NUCLEUS-ASSOCIATED FIBRILLAR BUNDLES

IN ADDITION TO MICROTUBULES

ANDREW J.POWELL, GEOFFREY W. PEACE, ANTONI R.SLABAS AND CLIVE W.LLOYDBiosciences Division, Unilever Research Colusorth Laboratory,Sharnbrook, Beds, U.K.

SUMMARY

Cytoskeletons prepared from carrot protoplasts often retain the nucleus, which is associatedwith bundles of 7 nm fibrils. These were also seen in cytoskeletons from oil palm, maize andItalian rye grass cells. By examining such cytoskeletons by immunofluorescence, negativestaining and stereo scanning electron microscopy it appears that the bundles could be part of amore delicate system of fibrils distal to the nucleus. Optical diffraction of the bundles, stainingwith heavy meromyosin, drug treatments and phalloidin-staining all confirm that the bundlesare not composed of actin. Bundles could not be isolated from such cytoskeletons in quantitiessufficient for analysis, but by disrupting protoplasts prepared from stationary-phase cells it waspossible to purify fibrillar bundles that appeared in the lysate. These were similar in size andproperties to the nucleus-associated bundles. The former were shown by sodium dodecyl8ulphate/polyacrylamide gel electrophoresis to be composed of several proteins and weresensitive to reducing agents. Since they are not composed of actin, the possibility was exploredthat these fibrils are related to the P-proteins of phloem sieve tube and accessory cells.

INTRODUCTION

Fibrillar proteins have long been known, by electron microscopy, to pervadecytoplasm but the advent of detergent-extraction techniques has led to a fuller apprecia-tion of the complexity and interaction of the component structural assemblies. Theadvantage of such techniques is that they afford a view of the skeletal, detergent-insoluble proteins in entire cells rather than in sections. In this way it has becomepossible to see that the cytoplasm of fibroblasts, for instance, is composed of threedifferent cytoskeletal systems (actomyosin, microtubules, intermediate filaments),each with a characteristic distribution. Using selective extraction it has been shownthat actomyosin and microtubles can be removed from fibroblasts to leave 'nuclearmonolayers' in which the nucleus is anchored by intermediate filaments (Osborn &Weber, 1977; Lehto, Virtanen & Kurki, 1978).

For plant cells, there is virtually no equivalent picture of the distribution of fibrillarproteins known from electron microscopy to be present. This is probably becausewall-less plant protoplasts do not conveniently display themselves by spreading uponthe substratum in the way that animal cells do. Instead, they sit high upon the supporting

320 A. J. Powell, G. W. Peace, A. R. Slabas and C. W. Lloyd

surface and consequently do not survive well the rigours of extraction; thefragility of enzyme-treated protoplasts and the presence of large, central vacuolesproviding added complications. However, it is known that protoplasts attached topolylysine-treated coverslips and then burst by a hyptonic shock leave behind disks ofadherent plasma membrane to which microtubules are attached in a detergent-resistant manner (Marchant, 1979; Lloyd, Slabas, Powell & Lowe, 1980). In orderto prepare more complete cytoskeletons, it was decided in this study to lyse adherentprotoplasts with detergent under isotonic conditions. Some cytoskeletons are formedin which the nucleus (previously absent in hypotonically lysed protoplsts) remains, inwhich case it is invariably associated with a bundle(s) of 7 nm fibrils. These fibrilsare the subject of this paper.

MATERIALS AND METHODS

Cell suspension cultures

Suspensions of non-embryogenic carrot cells (Daucus carota L. cv. Scarlet Nantes), originallyisolated from secondary phloem of roots, were maintained in Murashige & Skoog's medium(Gibco) containing 1 mg/1 2,4-dichlorophenoxyacetic acid, 0-5 mg/1 kinetin, 5 % (v/v) coconutmilk, 3 % (w/v) sucrose. Oil palm (Elais guineensit) suspensions were maintained in half-strength Murashige & Skoog's medium, supplemented with 2-5 % (w/v) sucrose, 2 mg/1 2,4-dichlorophenoxyacetic acid and 1 g/1 casein hydrolysate. Italian rye grass (Lolium pentaflorumL.) suspensions were maintained in Murashige & Skoog's medium containing 3 % (w/v)sucrose and 2 mg/1 2,4-dichlorophenoxyacetic acid. Maize (Zea mays L.) cultures were main-tained in the CC medium of Potrykus, Harms & Lorz (1979). The two latter cultures werekindly provided by Dr M.G. K. Jones (Welsh Plant Breeding Station, Aberystwyth, U.K.).

All cultures grow as fine suspensions and were maintained in conical flasks on a rotary shakerin a 12 h light/12 h dark cycle.

Protoplasts

In previous studies (Lloyd et al. 1980; Powell, Lloyd, Slabas & Cove, 1980) entire networksof plasma-membrane-associated cytoplasmic microtubules of carrot and moss cells and proto-plasts were stained with anti-tubulin antibodies. In order to ensure that these delicate arrayswere not perturbed by the withdrawal of plasma membrane away from the wall, plasmolysisof the cells (a routine step in preparing protoplasts) was avoided. It was also found to be import-ant to ' cushion' the protoplasts during incubation with enzyme by the addition of the minimumconcentration of sorbitol, which prevents swelling and bursting. To ensure that protoplastswere made from populations of actively dividing cells, cultures were sub-cultured 2 days priorto use.

Carrot cells were placed in 5 % (w/v) Onozuka R-10 cellulase (Kinki Yakult Mfg Co., 8-21Shingikan-Cho, Nishinomiya, Japan) for up to 4 h in growth medium containing 0-25 M-sorbitol. Protoplasts of oil palm cultures were prepared by treating cells with 4 % (w/v) Driselase(Fluorochem Ltd, Glossop, U.K.) in 0-3 M-sorbitol medium for 60-90 min. Rye grass cellswere placed in 2 % (w/v) Onozuka R-10 cellulase, 1 % Macerozyme (Kinki Yakult), 1 %Driselase, 1 % Rhozyme (Rohm & Haas, Philadelphia) in 0-7 M-sorbitol medium for 4$—5 h.Protoplasts of maize were prepared by treating cells with 1 % Onozuka R-10,0-5 % Macerozymeand 0-5% Rhozyme in o-6 M-sorbitol medium for 2 h.

Extraction of protoplasts with detergent

Copper electron microscoscope (EM) grids (200 mesh finder grids) were prepared by coatingthem with Formvar. They were then coated with carbon before being treated with a solution(1 mg/ml) of polylysine (Miles-Yeda Ltd) in distilled water. The protoplast pellet was sus-

Nucleus-associated bundle in plant cells 321

Fig. 1. Carrot protoplasts prepared without plasmolysis were extracted with TritonX-100 in isotonic MTSB. Under these conditions, nuclei are sometimes retained with-in the' ghost' or cytoskeleton. In A, a large, curvilinear structure can be seen by phase-contrast microscopy to be associated with the nucleus. Staining with a serum from anon-immunized rabbit, followed by FITC-goat anti-rabbit IgG, shows this structure(B) to connect the nucleus to a more diffuse pattern of staining at the protoplast'speriphery. X1125.

pended in an equal volume of medium and one drop of this suspension was placed onto eachpolylysine-coated grid for 20 min. Unattached protoplasts were then washed off by two rinsesin the medium used for their preparation (but without enzymes). Grids with adherent proto-plasts were then immersed in microrubule-stabilizing buffer (MTSB), 100 mM-PIPES (pH6-9), 2 mM-EGTA, 1 mM-Mg1+, to which sorbitol (equivalent to the concentration used in themedium for preparing protoplats) and 2-5 % (v/v) Triton X-100 had been added.

Negative stainingAfter 20 min the grids were briefly washed twice in this medium (but now without detergent)

and drained before being negatively stained by immersion for 20 s in a 1 % (w/v) aqueoussolution of uranyl acetate. They were then air-dried. The preparations were unfixed because


Fig. 2. Whole mount of negatively stained carrot cytoskeleton. The protoplast hadbeen attached to a polylysine-coated Formvar grid prior to extraction with detergent.In this example, an area of the protoplast's periphery is particularly well-preservedsince it contains microtubules (mi) known from previous studies to be associated withthe plasma membrane in a detergent-resistant manner. The nucleus (n) is also retainedand several fibrillar bundles (Jb) are seen to emerge from it and to terminate upon thesubstratum in the vicinity of the cortical microtubules. x 3500.

the use of glutaraldehyde or formaldehyde reduced the definition of microtubules and especiallyof fibrillar bundles. Samples were examined with a JEOL 100C transmission electron micro-scope.

Thin sectioningIn some cases, fibrillar bundles, which had been identified in these whole mounts, were

subsequently embedded in order to cut thin sections. This was achieved by recording theirpositions on finder grids so that, after flat-embedding in Spurr's resin, the cutting face of theblock could be aligned and trimmed to produce transverse sections. Transverse sections aretherefore derived from bundles that have been clearly established as being associated withthe nucleus in whole mounts.

Scanning electron microscopyThe same procedure was adopted as described above, except that glass coverslips were used

instead of grids and detergent-extracted cytoskeletons were fixed in 3 % glutaraldehyde (v/v)for 20 min prior to critical-point drying from acetone. Gold-coated samples were examinedusing a JEOL 35 scanning electron microscope.

Nucleus-associated bundle in plant cells

Fig. 3. Detail of a negatively stained carrot protoplast that had been extracted withdetergent under isotonic conditions. This shows a fibrillar bundle (Jb), distal to thenucleus to which it is attached, gub-dividing into a finer meshwork of protofibrils.The meshwork extends over the substratum in the same plane as plasma membrane-associated microtubules (mi), x 66000.

Indirect immunofluorescenceProtoplasts, attached to substrata, were prepared for indirect immunofluorescence as de-

scribed previously (Lloyd et al. 1979). The primary antibody was non-immunized rabbit serum,which was localized with fluorescein-conjugated (FITC) goat anti-rabbit, immunoglobulin G(IgG) (Miles-Yeda) at 1:3O dilution. To ensure that structures observed in the light microscopewere the same as those identified by electron microscopy, cytoskeletons were photographed onFormvar-coated finder grids and then re-located by negative staining in the EM.

Diffraction studiesIn detergent-extracted cytoskeletons, the nucleus-associated fibrillar bundles could be seen

by negative staining to have characteristic striations and these were further examined byoptical diffraction. Diffraction patterns were obtained from micrographs of negatively stainedmaterial in two ways. Principally, optical patterns were produced using a modified Rank-Pullendiffractometer (O'Brien & Bennett, 1972). For some of the micrographs, the image was con-verted to digital form and the Fourier transform was computed by Toltec Computer Ltd,Cambridge.

Heavy meromyosinProtoplasts were attached to grids and extracted in detergent-containing buffer in the standard

manner. Heavy meromyosin (HMM), prepared from rabbit skeletal myosin (Offer, Moos &Starr, 1973), was dissolved in 20 mM-NaCl, 1 mM-MgCl,, o-i mM-EGTA in 5 mM-phosphatebuffer (pH 7-0) at 0-5 mg/ml. The samples were treated with this solution, washed andnegatively stained. F-actin prepared from rabbit skeletal muscle (Offer, Baker & Baker, 1972)was attached to grids and was used in controls.


Fig. 4. Stereo-pair of critical-point-dried, detergent-extracted cytoskeleton of a carrotprotoplast viewed by SEM. A major bundle leaves the nucleus and as it approachesthe outer edge of the cytoskeleton it breaks down into thinner strands, x 6000.

Isolation of fibrillar bundles

Preliminary attempts to isolate nucleus-associated bundles in sufficient quantities for analysiswere unsuccessful. However, in related studies (Coventry, Hughes & Lloyd, unpublished) itwas found that if protoplasts were made from stationary-phase cells (6 days after a 10:100 sub-culture) instead of dividing cell populations (2 days after sub-culture), they produced freebundles of 7 nm fibrils when lysed with 2 % (v/v) Nonidet P40 (NP40), B.D.H. As will bediscussed, these are very similar, if not identical, to the nucleus-associated bundles of growingcells and were isolated as follows. The cells were converted to protoplasts by incubating themin 2% Driselase/i % Rhozyme (w/v), made up in sorbitol-containing medium, for z\ h at26 CC on a rotary shaker. They were washed three times, made into a thick suspension withI'S vol. buffer and lysed by the addition of NP40 to a final concentration of 2% (v/v). Thesuspension was gently agitated by passage through a Pasteur pipette and after being allowed tosettle for 15 min, a gelatinous precipitate containing nuclei was removed by filtration throughfour layers of muslin. Remaining nuclei, unbroken cells and large debris were removed fromthe filtrate by first centrifuging for 5 min at 250.gr and then centrifuging the supernatant ati(X>og over 1 M-sucrose for 7 min. Fibrillar bundles were then spun down from the supernatantat 22000g for 20 min at 10 °C. The pellet was resuspended in 1 ml of Dulbecco & Vogt'sphosphate-buffered saline (Flow Laboratories) and the bundles were then separated fromcontaminating vesicular material by density-gradient centrifugation using Percoll (PharmaciaLtd). For this the sample was layered on top of a 40 % (v/v) solution of Percoll in PBS andcentrifuged at 30000^ for 1 h. Bundles formed a band, which calibration studies with densitymarker beads showed to possess a buoyant density of approximately 1 -05 g/ml. That this bandwas highly enriched in bundles of 7 nm fibrils was confirmed by taking negatively stainedsamples for electron microscopy. Samples were also analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli (1970).


Fig. 5. Portion of a negatively stained fibrillar bundle attached (out-of-frame) to thenucleus in a carrot cytoskeleton. This shows that component 7 nm fibrils have alongitudinal periodicity that, in register with neighbouring fibrils, produces cross-striations. x 150000.

RESULTS

Light microscopy

During routine screening assays of antisera we obtained a rabbit serum that stainednucleus-associated cytoplasmic strands. The antigenic specificity of the serum is asyet uncharacterized but it provides useful information on the distribution of strands.Using this spontaneous antiserum, indirect immunofluroescence shows that indetergent-extracted protoplasts the nucleus is associated with linear structures(Fig. 1). By using finder grids for the antibody-staining of extracted protoplasts ithas been possible to re-examine these nucleus-associated elements in the electronmicroscope by negative staining with uranyl acetate (not shown). This indicates thatthe antiserum-stained bundles are identical with structures identified by other ultra-structural techniques described below. A double-coating of antiserum obscuresany fine detail but the linear structures are seen to be composed of bundles offibrils.

Negatively stained whole mounts

When carrot protoplasts prepared from growing cell suspensions are attached topolylysine-coated Formvar grids and extracted in isotonic buffer that contains de-tergent, nuclei frequently remain upon the substratum, a feature that we have neverseen when protoplasts are burst by hypotonic lysis. Using this latter method of'opening' the protoplasts, it has been shown (Marchant, 1979; Lloyd et al. 1980;Doohan & Palevitz, 1980; van der Valk, Rennie, Connolly & Fowke, 1980) that onlya circular fragment of adherent membrane remains on the substratum and this bearsmicrotubules. Such images are not eliminated by the present modifications but themore substantial cytoskeletons that also occur now contain the nucleus, which is

326

6A

A. J. Powell, G. W. Peace, A. R. Slabas and C. W. Lloyd

Fig. 6. In order to examine their internal arrangement, nucleus-associated fibrillarbundles were first visualized by negative staining (as in Fig. 5) before being flat-embedded in resin so that they could be sectioned at right angles to their long axes.In A, the component fibrils can be seen as separate elements although there is asuggestion of cross-bridging. The more usual appearance, however, is demonstratedin B, in which the bundle is composed of fibrils arranged in several plies, x 225 000.

Table 1. The stability of fibre bundles in carrot protoplasts to various treatments

TreatmentBundlespresent?

24 h at o °C»o-6 M-KIfCytochalasin B, 24 h, 10 fig/m\*Colcemid, 24 h, io~* M #

Colchicine, 24 h, io"8 M#

Ionophore A23187 (10/ig/ml) plus 10 mM-Ca1+, 5 h*DNase type I, 5 h, 10 fig/ml and 200 /tg/mlfTrypsin, 100 fig/ml, 1 h, 25 °CfPronase, 100 fig/m\, 1 h, 25 °CfDithiothreitol, 0-2 % (w/v), 30, 60 or 120 min, o °CfCaffeine, 05 % (w/v), 1 h#

Homogenization of protoplasts in:(a) Isotonic microtubule-stabilizing buffer (MTSB)(6) Isotonic MTSB plus 2-5 % (v/v) Triton X-100(c) Isotonic MTSB plus 1 % (v/v) Sarcosyl

++++++

++

* Protoplasts were pre-treated by adding the agent to the protoplasting medium prior toextraction with detergent.

t Detergent-extracted cytoskeletons, as opposed to protoplasts, were treated with theseagents.

J Fibre bundles still visible on grids by negative staining but the structure is perturbed.

Nucleus-associated bundle in plant cells

Fig. 7. Optical diffraction pattern from part of a negatively stained, nucleus-associ-ated fibrillar bundle. In Fig. 5, the cross-striations on bundles were illustrated and thisdiffractogram is from a similar photograph. The pattern shows two meridionalreflections: the first and second order of an axial repeat of i /y6 nm and a layer lineat 1/22 nm-1.

invariably associated with one or more fibrillar bundles. These bundles vary in lengthfrom 5 to 17 fim.

In a well-preserved cytoskeleton (Fig. 2) several bundles can be seen associatedwith the nucleus. Distal to the nucleus these bundles are often seen to sub-divide.In Fig. 3, for instance, a bundle breaks down into individual fibrils that run parallelto the substratum. This region is probably the sub-membranous cell cortex since itcontains microtubules, known from previous work (Lloyd et al. 1980) to be associatedwith the substratum in a detergent-resistant manner. Stereoscopic examination ofcritical-point-dried cytoskeletons confirms the impression derived from these air-dried whole mounts, that the nucleus is connected by the fibre bundles to the regionadjacent to the substratum (Fig. 4).

In the negatively stained preparations, however, the bundles have a characteristicappearance, in that they are somewhat striated due to lateral register of the componentfibrils (Fig. 5). Some of these bundles were subsequently embedded in resin andtransverse sections were cut normal to the substratum. Fourteen bundles weresectioned in this way, all of them being nucleus-associated fibrillar bundles alreadyexamined in whole mounts. The predominant picture was of fibrils joined in sheets,the cross-section of the structure being composed of several such plies (Fig. 6). Thatthe orientation of these plies was not a compression artefact generated by sectioningwas suggested by the fact that they were observed to run at right angles to the sub-stratum (i.e. at right angles to the cutting edge of the knife) as well as parallel to it indifferent bundles within the same section. In only one instance was the bundle seen to


8 MFig. 8. When protoplasts from dense cell suspensions (unlike the freshly sub-culturedcells in previous figures) are lysed in detergent, bundles of 7 nm fibrils assemble inthe supernatant. They are similar in appearance, in lack of staining with FITC-phal-loidin and in sensitivity to reducing agents, to the nucleus-associated bundles ofsubstrate-attached cytoskeletons. It is these' supernatant' bundles that are analysed bySDS-PAGE. Negatively stained with methylamine tungstate. x 150000.

be composed of individual fibrils, in which case they seemed, in places, to be con-nected by cross-bridges (see Fig. 6 A).

Similar nucleus-associated bundles were observed in detergent-extracted proto-plasts derived from oil palm suspension cells as well as in Italian rye grass and Zeamays suspensions.

Stability of cytoskeletal components

To help characterize them, the fibrillar bundles were subjected to various treat-ments known to perturb cytoskeletal structures; these are summarized in Table 1.Wherever possible, the treatment was performed on protoplasts, but where access toenzymes was required the protoplasts were made permeable with detergent.

Both colchicine and colcemid in millimolar concentration depolymerized micro-tubules, but with both these agents the nucleus remained within the extracted cyto-skeleton, as did their associated fibre bundles.

To test the possibility that the bundles might be composed of actin, protoplastswere treated for 24 h in 10/Jg/ml of cytochalasin B. This had no effect on the in-tegrity of the bundles. Both the detergent Sarcosyl and o-6 M-KI perturbed thebundle, causing the fibrils to be less well-ordered, but they did not cause depoly-merization. Trypsin was without effect but Pronase and the reducing agent dithio-threitol caused the bundles to disappear. This suggests that the stability of theseproteinaceous, nucleus-associated fibrillar bundles is due, at least in part, to thepresence of S-S bonds.


A B C

Fig. 9. The proteins of a fibrillar bundle preparation as analysed by SDS-PAGE. Itwas not possible to purify nucleus-associated fibrillar bundles, in quantity, fromcytoskeletons of recently sub-cultured cells. However, it was found that when culturesaged and entered stationary phase, that free-floating bundles of 7 nm fibrils polymer-ized in the detergent lysate of protoplasts prepared from those cells. As discussed inthe text, these latter bundles are similar in several respects to nucleus-associatedbundles observed in cytoskeletons. Lane A contains molecular weight markers at 94,68, 53, 21 and 14 (x 10s). Fibrillar bundles were isolated from Percoll-densitygradients and centrifuged at 20000 # to form a pellet. By comparing the gel pattern ofthe bundle-containing pellet (lane B) with the supernatant (lane c) it will be seen thatthe predominant band in the former occurs at 62 x io* M, and that other bands, ex-clusive to the pellet, occur at 125, 65 and 30 x io*Mr. The pellet is also enriched witha doublet at about 15 x io3 Mt.

Heavy meromyosin

To test the possibility that the bundles were composed of F-actin, they werechallenged with heavy meromyosin prepared from rabbit skeletal muscle. Filaments ofF-actin were also tested on parallel, control grids. The HMM successfully formedarrowheads in the latter case but was never seen to do so with the nucleus-associatedbundle. It is conceivable that HMM could be sterically excluded from an actinparacrystal by the presence of an actin-binding protein, as Tilney (1975) has demon-strated for the crystalline actin inclusion in Limulus sperm. However, where the plantbundles were either snapped and frayed or dispersed into well-separated, individualfibrils upon the substratum, they were not decorated by HMM. In addition, they donot stain with an FITC conjugate of the F-actin-binding metabolite, phalloidin(obtained from Dr J. Wehland), at concentrations that stain stress fibres in fibroblasts.


Diffraction studies

The strongest and most consistent feature of the diffraction pattern (Fig. 7) frommicrographs of negatively stained bundles is a meridional or near-meridional re-flection whose axial spacing corresponds to 73 nm (average of 5). This confirms thepresence of a regular, approximately axial repeat in the bundles. In patterns fromregions of the fibres where the fibrils appear to be well-aligned, an equatorial spacingof 7-10 nm is observed. In addition, there are reflections, usually weak, lying on layerlines whose axial spacings are orders of approximately 3 x 7-3 nm (22 nm). Thepresence of these reflections may suggest that the fibrils have helical symmetry. Wehave not been able to determine the structure more precisely because of disorder inthe bundles, which presumably occurs during the preparation for microscopy. How-ever, no reflections characteristic of other fibrous cytoskeletal structures, such asmicrotubules and F-actin, have been detected.

Isolation of fibrillar bundles

Bundles of 7 nm fibrils are purified from detergent-lysed suspensions of protoplastsprepared from cultures in stationary phase. The bundles are several micrometres inlength and their isolation can be monitored at all stages by light microscopy andnegative staining (Fig. 8). Following differential (and Percoll-density) centrifugation,a fraction is obtained, which (as judged by negative-staining) contains only fibrillarbundles free of organellar contamination. These were centrifuged out at 20000^ toform a pellet and both pellet and supernatant were analysed by SDS-PAGE underreducing conditions. The bundles are depolymerized by dithiothreitol or by mercepto-ethanol. The predominant band in the pellet (Fig. 9) has an apparent molecularweight (Mr) of approximately 62 x io3. Compared to the supernatant, the pellet isalso enriched with a low molecular weight doublet at about 15 x io3 MT. Other bandsat approximately 30 and 125 x io3 MT are exclusive to the bundle-containing pellet.

DISCUSSION

Distribution of fibrillar bundles in protoplasts

The present results demonstrate a system of fibrils that is associated with the nucleusin detergent-extracted protoplasts derived from both monocotyledonous and dicotyle-donous cell suspensions. In these substrate-attached cytoskeletons the bundles areinvariably attached to the nucleus but their distribution distal to the nucleus is not soclear and this is probably a reflection of the way in which they are prepared. Forinstance, the bundles once extracted are presumably brittle, for snapped ends arecommonly seen. Unlike animal tissue-culture cells, which actively develop a' flattened'cytoskeleton as a part of the process of spreading upon the substratum, plant proto-plasts retain an essentially spherical shape.

Some of these protoplasts, if not all, will already have undergone a change of shapeduring their preparation from non-spherical cells, but the distribution of cytoskeletal


elements is likely to be perturbed further when the supportive central vacuole iscollapsed by detergent treatment and the residual material is dried down. There are,therefore, technical barriers to presenting a native distribution of cytoskeletal elements.Nevertheless, there are cases where the nucleus-associated bundles are not snapped.In Fig. 3, for instance, several bundles emerge from a nucleus and sub-divide intofiner bundles as they approach the substratum, where well-preserved cortical micro-tubules are to be seen. In Fig. 4, a bundle frays into individual fibrils upon the sub-stratum where, again, microtubules (which are known from previous work to beattached to the plasma membrane in a detergent-resistant manner; Lloyd et al. 1980)are found. This suggests that fibrils could be bundled when close to the nucleus butare dispersed in the cortical regions of the cell, distal to the nucleus. If so, the fibrilswould be more delicate in the un-bundled form and, as it seems, do not often survive.That the linear bundles connect the nucleus to the cell periphery is also suggested bythe immunofluorescent staining in Fig. 1. The fact that the serum used for thoseexperiments is as yet uncharacterized should not detract from this observation; theserum is used here only as a convenient non-specific stain. However, by recording theposition of an antibody-stained fibrillar bundle, using the light microscope, it ispossible, following negative staining, to re-locate the same component in the electronmicroscope. Fine detail is obscured by the double coating of antibody (and for thisreason is not shown), but it is possible to confirm that the linear elements viewedunder fluorescence optics consist of bundles of fibrils. These are likely to be the samefibrillar bundles observed without staining with antiserum, for no other structure ofthat size, with which they could be confused, has ever been seen in cytoskeletons.

These observations establish that bundles of fibrils, several micrometres in lengthare associated with the nucleus of cytoskeletons prepared from carrot suspension cells.Similar observations have been made with oil palm, Italian rye grass and maize cellsuspensions.

As for their size, the fibrils are about 55 run in diameter in sections prepared fromnegatively stained bundles in which the average diameter had already been calculatedas 7-3 run. The sectioned and dehydrated material has therefore shrunk and for thisreason the diameter of native fibrils might be expected to lie closer to the 7 nm value.This value agrees well with the equatorial reflections in optical diffractograms ofnegatively stained, un-sectioned fibrillar bundles.

The question of artefacts

In discussing the morphology and distribution of these components it is importantto consider the question of artefacts since, because of their paracrystalline nature,there is a prima facie case that they appear only during experimentation. Similar ifnot identical bundles have, however, been encountered in sections of fixed andembedded oil callus (unpublished) and have already been reported in thin sections ofcarrot suspension cells by Wilson, Israel & Steward (1974). In that case 'fibrillarbundles' (to use their terminology) are seen in the cytoplasm close to the nucleus andare about 0-25 /im in diameter and several micrometres in length. Withers (1980) hasalso reported bundles of similar dimensions in thin sections of glutaraldehyde-fixed


carrot suspension cells. There is good reason to believe, then, that fibrillar bundles ofthe kind detected in detergent-extracted carrot cytoskeletons exist in fixed andembedded carrot cells, in which case their formation would not appear to be a by-product of our experimental treatments.

There are many other reports of bundles of fibrils within plant cells (e.g. see O'Brien& Thimann, 1966; Thomas, Konar & Street, 1972; Parthasarathy & Muhlethaler,1972; Parthasarathy & Pesacreta, 1980). Collectively, these suggest that unidentifiedbundles of 5-7 nm microfilaments commonly occur in the cortical cytoplasm andtransvascuolar strands of elongating cells from dicotyledonous and monocotyledonousplants.

Identity of fibrillar bundles

In examining precedents for cytoplasmic fibrils there are perhaps two major candi-dates: actomyosin and P-protein.

Actomyosin. The paracrystalline bundles identified here do bear a superficial re-semblance to actin paracrystals. In cross-section, for instance, the laminated appear-ance recalls actin bundles examined by Tilney (1975) in the false discharge of horse-shoe crab sperm and in the stereocilia of the alligator lizard's cochlea (Tilney, DeRosier & Mulroy, 1980). In Limulus the bundles did not bind heavy meromyosin butdid so when microfilaments were reconstituted from an acetone powder. Lack ofdecoration by HMM (in that case due to steric exclusion caused by the presence of anactin-binding protein) does not, therefore, contra-indicate actin, as our inability todecorate plant bundles (as opposed to individual fibrils) might initially imply. How-ever, it should be noted that single fibrils that frayed out from the bundles did notbind HMM either. Neither does the insensitivity of the bundles to cytochalasin Bnecessarily rule out actin since this drug has actually been reported to promote theappearance of microfilament bundles in the cytoplasm of maize coleoptile cells(Cande, Goldsmith & Ray, 1973). That the fibrillar bundles are not composed of actinis implied by three lines of evidence. The first is that F-actin, whether alone or in thepresence of additional components, always gives rise to reflections in the diffractionpattern characteristic of its double-stranded, beaded structure; whether muscle thinfilaments (F-actin, tropomyosin, troponin; O'Brien, Bennet & Hanson, 1971) or theacrosomal process of Limulus (F-actin, 55 x io3 MT actin-binding protein; De Rosieret al. 1977). Such layer lines at approximately 36, 59 and 5-1 nm are subject toshrinkage (Tilney et al. 1980) but even so, it is difficult to construe any obvious rela-tion with the 7-3 nm and the fainter 22 nm layer lines that are detected in this presentstudy. That plant actin might be expected to share the packing characteristics of actinfrom other sources such as these (and hence not represent some unusual form) issuggested by the work of Palevitz & Hepler (1975), who found a repeat distance ofabout 36-37-5 nm for negatively stained paracrystalline bundles of Nitella, whichapproximated to the 38 nm repeat for the arrowhead decorations of HMM. Opticaldiffraction patterns of the HMM-decorated bundles in Vallisneria leaves are alsopurported to share common features with patterns from skeletal muscle actin (Yama-


guchi & Nagai, 1981). It may be relevant to note that in these cases where actin micro-filaments were positively identified in plant cells no mention was made of any inter-action with the nucleus, although they are seen to be closely associated with chloro-plasts. The second line of evidence against these carrot bundles being composed ofactin derives from the analysis of isolated bundles. SDS-PAGE profiles of fractionscontaining fibrillar bundles show enrichment of a 62 x io3 MT and other proteins,whereas no band was found to co-migrate with skeletal muscle actin. Thirdly, thebundles did not stain with rhodamine (or FITC)-phalloidin, which is known to bindF-actin (Wehland, Osborn & Weber, 1977).

P-protein. When considering paracrystalline or fibrillar inclusions (not composed ofactomyosin) in plant cells, it is necessary to touch upon P-protein (Esau & Cronshaw,1967), a term applied to aggregations of protein encountered in phloem sieve, paren-chyma and companion cells. Ultrastructurally, P-proteins are polymorphous and, toquote Cronshaw (1975), 'are granular, tubular, fibrillar or crystalline depending onthe species or the maturity of the cells in which they are observed'. This variabilityin P-protein morphology is also reflected in the fact that, for the fibrillar form, thefibril diameter can, depending on species, vary from about 4 to 20 nm (Sabnis & Hart,1982). In their analysis, too, there is an equally wide variation in reported molecularweights for proteins obtained from different phloem exudates. Sabnis & Hart (1982),for instance, set the limits of the range as far apart a s iox io 3 and 130 x io3 Mr. Theredo not seem, therefore, to be any generally agreed characteristics that would helpcategorize fibrillar proteins in non-phloem cells (i.e. suspension cultures) as beingP-protein. Nevertheless, there is little in the present observations to contra-indicatethat the fibrillar bundles belong to the P-protein family; indeed there are severalconsistent features.

For instance, Hepler & Palevitz (1974) characterize P-protein as exhibitingmolecular weights in multiples of 15 x io3, being soluble in reducing agents andpolymerizing into filaments in o-i M-KC1. We were unable to isolate nucleus-associated fibrillar bundles from cytoskeletons of growing cells in suitable quantitiesfor analysis. Instead it was found that fibrillar bundles polymerized into suspensionwhen protoplasts from stationary-phase cells were lysed. Like the nucleus-associatedbundles, these were composed of 7 nm fibrils, were sensitive to reducing agents anddid not stain with FITC-conjugated phalloidin. When subjected to SDS-PAGE underreducing conditions, the lysate bundles yielded bands in approximate multiples of15 x io3 (15, 30, 62, 125), which correspond to the pattern of P-proteins from phloemexudates (Weber & Kleinig, 1971; Beyenbach, Weber & Kleinig, 1974; Nuske &Eschrich, 1976). The fibrils in some sieve-tube exudates are also 7 nm in diameter.It is proposed, therefore, that the lysate fibrillar bundles could be related to P-proteinsand that the nucleus-associated bundles are another form of the same protein(s). Thisis being studied further.

As for the role of these fibrils, it seems unlikely that in suspension cells they haveany function related to normal phloem physiology, since it is reported that evenislands of phloem cells within carrot callus do not translocate photosynthetic materials(Hanson & Edelman, 1970). But even in fully functional phloem the role of P-protein


remains unclear, which presents great difficulty in assigning functions to ostensiblysimilar fibrils in suspension cells. What does seem clear, though, is that in substrate-attached, detergent-extracted cytoskeletons prepared from cultures of growing cells,there are bundles of 7 nm fibrils attached to the nucleus. However, it has beenargued (Walker & Thaine, 1971) that because of their chemical properties, phloemP-proteins are altered by fixation and by experimental conditions that favouroxidation, and that this seriously hinders interpretation of their distribution in theliving state. A nucleus-anchoring rdle for the present fibrils remains an attractivepossibility, but in view of these considerations we must await the outcome of furtherwork before accepting this conclusion.

We thank Dr Pauline Bennett, MRC Cell Biophysics Unit, King's College, University ofLondon for help and advice with optimal diffraction; Dr Gerald Offer of King's for supplyingthe HMM; Dr Roger Craig of the MRC Laboratory of Molecular Biology, Cambridge, U.K.for advice on using HMM; Chris Clay for assistance with the SEM and Christine Evans forhelping with the photography. We also thank Dr W. A. Hughes and Ruth Coventry for sharingsome recent findings.

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(Received 25 October 1981 - Revised 18 January 1982)

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THE DETERGENT-RESISTANT CYTOSKELETON OF ...Fibrillar proteins have long bee by electron knownn microscopy, to pervad, e cytoplasm but the advent of detergent-extraction techniques