light-microscopic observations of individual microtubules … · 2017-06-25 · reconstituted...

J. Cell Sci. 19, 607-620 (1975) 607

Printed in Great Britain

LIGHT-MICROSCOPIC OBSERVATIONS OF

INDIVIDUAL MICROTUBULES

RECONSTITUTED FROM BRAIN TUBULIN

RYOKO KURIYAMA AND TAIKO MIKI-NOUMURADepartment of Biophysics and Biochemistry, University of Tokyo,and Institute of Molecular Biology, Nagoya University, Nagoya, Japan

SUMMARYThe course of polymerization of individual brain microtubules could be observed with a

light microscope employing dark-field illumination. Statistical analysis of the increase in micro-tubule length during the polymerization was in accordance with the time course of viscositychange of the tubulin solution. After a plateau level in viscosity was attained, there was no sig-nificant change in histograms showing length distribution. These observations were confirmedwith fixed and stained microtubules, using a phase-contrast microscope.

Observations with dark-field illumination revealed that reconstituted microtubules depoly-merized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydrylreagents such as £-chloromercuriphenyl sulphonic acid (PCMPS) and ^-chloromercuribenzoicacid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules whichwere assembled by mixing purified tubulin dimers with ciliary outer fibres could also be fol-lowed with these optical systems.

INTRODUCTION

The microtubules which are found in various kinds of organisms are supposed totake part in cellular motility, transportation of cellular constituents or maintenance ofcell shape. In order to make clear the functions of cytoplasmic microtubules in the cell,it seems requisite to clarify the details of microtubule polymerization and depoly-merization.

In 1972, Weisenberg (1972) demonstrated the reconstitution of brain microtubulesin vitro; this work has been confirmed by several groups of investigators. In all ofthese studies, the process of microtubule assembly was analysed by electron-microscopic observation and by physicochemical methods, such as measurement ofviscosity (Olmsted & Borisy, 1973; Kuriyama & Sakai, 1974; Haschke, Byers &Fink, 1974), flow birefringence (Haga, Abe & Kurokawa, 1974) or turbidity(Shelanski, Gaskin & Cantor, 1973; Houston, Odell, Lee & Himes, 1974; Gaskin,Cantor & Shelanski, 1974).

Macnab & Koshland (1974) observed individual flagella of Salmonella in the nativestate under a light microscope using dark-field illumination. Recently, Hotani (1974)succeeded in visualizing individual flagella of Salmonella and photographing them byusing a light microscope of higher efficiency equipped with dark-field optics. Employ-ing a similar optical system and technique for micrography, a reversible morphological

608 R. Kuriyama and T. Miki-Noumura

transformation of the bacterial flagellum during pH changes was demonstrated byKamiya & Asakura (1974).

Although Summers & Gibbons (1971) have shown dark-field micrographs ofaxonemes or of doublet microtubules from sperm tails, a singlet microtubule has notyet been observed. Since its diameter (about 24 nm) is greater than that of theSalmonella flagellum (15 nm), it was suggested that singlet microtubules ought to bevisible with dark-field illumination. This was first achieved with rabbit brain micro-tubules by Miki-Noumura and Kamiya (manuscript in preparation), who found thatthe reconstituted singlet microtubule was quite straight.

In the present paper, we report observations of native microtubules reconstitutedfrom porcine brain tubulin, made with dark-field optics, and of silver-stained micro-tubules observed with a phase-contrast microscope. The process of tubulin poly-merization was analysed by measurements on photographs of the average lengths ofreconstituted microtubules in comparison with the viscometric change duringpolymerization. Besides the homogeneous microtubules of brain tubulin, the processof assembly of heterogeneous microtubules derived from ciliary outer fibres ofTetrahymena and brain tubulin dimers was also monitored with light microscopy.

MATERIALS AND METHODS

A crude tubulin fraction (50000 g supernatant of brain homogenate), a partially purifiedtubulin fraction (recovered after 1 cycle of polymerization and depolymerization), and atubulin dimer fraction (trailing fraction obtained by Sephadex G-200 gel nitration of thepartially purified tubulin fraction) were prepared as described elsewhere (Kuriyama & Sakai,1974; Kuriyama, 1975). A purified tubulin dimer preparation was also obtained by lineargradient elution of the partially purified fraction through a DEAE-Sephadex A-50 column orby discontinuous elution according to the method of Weisenberg, Borisy & Taylor (1968),except that the elution medium consisted of 0-3 M or o-6 M KC1 in 5 mM MES, 0 5 minMgSOa, 10 mM EGTA and i-o mM ATP (pH 67) . The reassembly buffer consisted of 50 mMor 100 mM KC1, 0 5 mM MgSO4, i-o mM EGTA, i-o mM ATP, 5 mM MES (2-(iV-morpho-lino)ethanesulphonic acid)-KOH buffer (pH 67) .

Ciliary outer fibres were obtained from Tetrahymena pyriformis by the methods of Gibbons(1965) and Stephens & Levine (1970), with some modifications. The axoneme fraction wasdialysed overnight against 200 vol. of 1 mM Tris-HCl, 0 1 mM EDTA, o-i mM dithiothreitolat pH 8o , followed by centrifugation at 40000 g for 30 min. The pellet was resuspended anddialysed overnight against the same solution. After centrifugation, the outer fibre pellet wassuspended in an ATP-free reassembly buffer containing 6 M glycerol, and stored at -20 °C.

The optical system consisted of an Ushio 100-W mercury arc lamp, a Nikon SUR-K typemicroscope equipped with Olympus DC condenser (N.A. 1-20-1-33), an Olympus apochro-matic objective lens (40 x , oil immersion type, N.A. i-o), a Nikon 10 x eye piece and a NikonEFM camera. Photographs were taken on Kodak Tri-X Pan film with an exposure of 2 s.The effective speed of the film was increased to ASA 3200 by Pandol developer (Fuji PhotoFilm Co.).

The silver nitrate method is a technique commonly employed to visualize bacterial flagellaunder the light microscope (lino, Suzuki & Yamaguchi, 1972). Fixation and staining of micro-tubules were done following the method of Nishizawa & Sugawara (IkagakukenkyujoGakuyukai, 1958), with some modifications. One drop of sample was put on a well cleanedslide, which was then immersed in fixing solution (4% tannic acid, o-oi % FeCls, 5 %glutaraldehyde, 0 0 0 8 % NaOH) for 10-15 rnin, thoroughly rinsed with distilled water, andstained with 4 % silver nitrate in aqueous ammonia. After it became brownish, it was washedagain with distilled water and dried. The silver nitrate solution gave satisfactory results over a

Reconstituted microtubules from brain tubulin 609

wide range of concentrations, from 0-1-10%. In some cases, the samples on the slides werepostfixed with fuchsin solution (mixture of 1 vol. of saturated fuchsin in ethanol and 10 vol.°f 5 % phenol) for 1-2 min to give better contrast. The preparations were observed under aphase-contrast microscope, Nikon SUR-K type. Photographs were taken at a magnification of1000 with a 1-8 exposure on Minicopy film.

Protein concentration was determined by the method of Lowry, Rosebrough, Farr & Randall(1951), using bovine serum albumin as the standard.

Viscometric measurement of tubulin polymerization and electron-microscopic observationswere performed as reported previously (Kuriyama & Sakai, 1974).

RESULTS

Observation of reconstituted microtubules with a light microscope

When a partially purified tubulin preparation containing about 2-6 mg/ml oftubulin was incubated at 35 °C in the reassembly buffer or in buffer containing4 M glycerol, microtubules were reconstituted, causing an increase in viscosity asdescribed previously (Kuriyama & Sakai, 1974; Kuriyama, 1975). After the viscosityreached a maximum value, the fraction was diluted with 100-200 vol. of reassemblybuffer containing 4 M glycerol (glycerol reassembly buffer) to permit clear observationof individual microtubules. It was confirmed that addition of glycerol to the reassemblybuffer was the most effective way of preventing depolymerization of the recon-stituted microtubules upon dilution.

Fig. 4 shows dark-field photographs of microtubules reconstituted from such apartially purified tubulin fraction in glycerol-free reassembly buffer at intervals afterincubation at 35 °C. Observations on fixed and stained microtubules were carriedout using reconstituted microtubules diluted with glycerol reassembly buffer or withbuffer containing 2-5% glutaraldehyde. After being stained by the silver nitratemethod, the reconstituted microtubules were observed under a phase-contrastmicroscope (Fig. 5). Each line observed in both figures is thought to correspond toone singlet microtubule, since the diameter of ciliary outer doublet microtubulesobserved with dark-field or phase-contrast microscopy was seen to be evidently widerthan the thickness of the lines in Figs. 4, 5 (data not shown). In order to make clearerthe point that each line was equivalent to an individual microtubule, the total numberand length of lines found in an aliquot of diluted sample were measured with boththe light microscope and the electron microscope. The results of a series of suchdeterminations indicated that the number and length distribution of lines obtainedwith the light microscope agreed with those of microtubules counted and measuredwith the electron microscope, within an error of 10-30 %. Therefore, the lines observedlight-microscopically can justifiably be identified as individual microtubules, and willbe called microtubules in this paper.

In the observations under the dark-field microscope, the polymerization processcould be followed on a slide by keeping the temperature of the mechanical stageconstant at 35 °C. Such unfixed microtubules depolymerized and disappearedimmediately when a solution of 2-7 mM ^-chloromercuriphenyl sulphonic acid(PCMPS), 0-5 mM />-chloromercuribenzoic acid (PCMB) or 5 mM CaCl2 dissolvedin glycerol or glycerol-free reassembly buffer was added from one side of the cover-


slip. Polymerized microtubules were also sensitive and responded readily to lowertemperature during observation. Details of these observations will be described in asubsequent paper. On the other hand, the microtubules fixed with glutaraldehydeshowed no tendency to depolymerize even after immersion for longer periods inreassembly buffer containing PCMPS, PCMB or CaCl2.

Correlation between length distribution of microtubules and viscosity increment duringpolymerization

When partially purified tubulin was incubated at 35 °C, the viscosity of the solutiongradually increased with increase in the time of incubation (Fig. 1 A). In parallel withthe viscometric measurements, observations with dark-field illumination were carriedout. At intervals after the beginning of incubation, aliquots of the tubulin fraction

a- 0-6 -

10 15 20 25Incubation time, mm

30

Fig. 1. A, viscosity increase in the course of tubulin polymerization. o-6 ml of partiallypurified tubulin fraction (2-6 mg/ml) was incubated at 35 °C in glycerol-free reassemblybuffer at zero time. Arrows indicate observation times under dark-field (see Fig. 4)and phase-contrast (see Fig. 5) microscope. B, mean lengths, in fim, of reconstitutedmicrotubules measured on the dark-field contrast photographs of Fig. 4. c, meanlengths, in /im, of reconstituted microtubules measured on the phase-contrastphotographs of Fig. 5.


were diluted with glycerol reassembly buffer and observed under the microscope.The results of a statistical analysis on a series of the photographs as shown in Fig. 4are summarized in Fig. 2. The viscosity change was in good agreement with thehistograms shown in Fig. 2; that is, the increase in viscosity was closely correlatedwith the increase in average length of the reassembled microtubules (Fig. 1 B). Afterthe plateau level of viscosity was attained there was no appreciable change in thelength distribution.

120

80

40

^ 40

2 20o

I 40

- B

o 20

40

20

- C

- D

60

40

20

60

40 "5

20 3S

20

40

20

0 12 24 36 48 60

Length, ftm

Fig. 2

72 0 12 24 36 48 60 72Length, fim

Fig. 3

Fig. 2. Diagrams of length distribution measured on the photographs of Fig. 4.Dotted line indicates average length of microtubules (see Fig. 1 B). A, B, C and D are at5, 10, 20 and3omin, respectively, and numbers of microtubules used were 267, 254,260 and 240.Fig. 3. Diagrams of length distribution measured on the photographs of Fig. 5.Dotted line indicates average length of microtubules (see Fig. 1 c). The numbers ofmicrotubules used for analysis for A, B, C, D respectively were 230, 282, 242 and 274.

These results were further confirmed by phase-contrast microscopic observation ofthe silver-stained microtubules. Photographs of the stained microtubules at eachincubation period are shown in Fig. 5. The length distribution of the stained materialsmeasured on these photographs is summarized in Fig. 3. The increase in averagelength of the reassembled microtubules during incubation also agreed well with theincrease in viscosity change in this case (Fig. 1 c).

It should be mentioned, however, that the mean length of reconstituted micro-tubules and the pattern of the length distribution were different when differenttubulin preparations were used. Furthermore, storage of tubulin fraction significantlyaffected the time course of tubulin polymerization and maximum length of recon-stituted microtubules obtained subsequently.

In contrast to microtubules from the partially purified tubulin fraction in glycerol-free reassembly buffer, the mean length of microtubules reconstituted from the


partially purified tubulin fraction in glycerol reassembly buffer was around io/tm,even after a 210-min incubation (data not shown). When a crude tubulin fraction wasincubated at 35 °C, longer microtubules of nearly 100/tm could be reconstituted(Kuriyama & Sakai, 1974). The viscosity increase of the preparation reached aplateau level within 10-20 min. The average length of reassembled microtubules fromthe crude tubulin fraction increased in parallel with the viscosity increment, asreported in the previous paper (Kuriyama & Sakai, 1974).

Observation of heterogeneous microtubules reconstituted from brain tubulin dimers andciliary outer fibres

Further experiments on heterogeneous microtubule reassembly were performedemploying the optical systems described above. Measurement of viscosity andobservation with electron microscopy suggested that the purified tubulin dimers areunable to reassemble into microtubules by themselves, and that fragments of micro-tubule are required as nuclei to induce the polymerization of tubulin (Kuriyama, 1975).Ciliary outer fibres were therefore used as nuclei in the present experiment. Althoughthe total length of the outer fibres from Tetrahymena cilia was about 5 /tm, fragmentsless than 2-3 /tm in length were obtained by dialysing the axoneme fraction for alonger period. Such a treatment allowed A- and B-tubules of the fragments to remainalmost intact, as shown in Fig. 7E. When purified tubulin dimers in glycerol re-assembly buffer were incubated with such outer fibres at 35 °C, heterogeneousmicrotubules were reconstituted from one or both ends of the outer fibres as shownin Fig. 6 A, B, E and F, and Fig. 7A-C. The length of the heterogeneous microtubulesbecame greater in parallel with the incubation time at 35 °C. Because of side-by-sideassociation among the outer fibres in reassembly buffer, reconstituted microtubulestended to form clusters or radiating structures as shown in Fig. 6 c, D, G and H, andFig. 7D and F.

Similar results were obtained with a tubulin dimer fraction in glycerol reassemblybuffer purified by DEAE-Sephadex A-50 column chromatography. The viscosity ofthis fraction, which was confirmed to contain only tubulin dimer, showed a slighttendency to increase. The addition of a small amount of partially purified tubulin orciliary outer fibre preparation induced a striking increase in viscosity. On chromato-graphy, a fraction eluted at 0—0-3 M KC1 contained a main component of highmolecular weight, which also promoted tubulin polymerization (data not shown).In all of these cases, numerous reconstituted microtubules were identified at thelight-microscopic level.

As demonstrated in Fig. 7, observation with electron microscopy revealed thatpolymerization of brain tubulin took place only from the A-tubule of ciliary outerfibres, which agrees with the report by Borisy, Olmsted, Marcum & Allen (1974).The length of the reconstituted microtubules formed in each incubation period inglycerol reassembly buffer was as follows: less than 1 /tm during 30 min, 5-6/tmduring 3 h. After overnight incubation, much longer microtubules were formed. Ata tubulin dimer concentration of 2 mg/ml, microtubules were reconstituted from both


ends of the outer fibre fragments. These microtubules reconstituted from both endsof an outer fibre were usually of unequal lengths.

DISCUSSION

The visualization of bacterial flagella by the use of a high-intensity arc lamp andefficient optical systems (Macnab & Koshland, 1974; Hotani, 1974; Kamiya &Asakura, 1974), suggested the possibility of identifying individual microtubules in anunfixed state at the light-microscopic level. This was done by Miki-Noumura andKamiya (in preparation), who found that reconstituted rabbit brain microtubules hada straight form. The same is true of the porcine brain microtubules used in the presentwork. On the other hand, partially trypsin-digested axonemes of sperm tails exhibiteda coiled form, having a diameter of about 3 /im, as reported by Zobel (1973).

The present observations using dark-field illumination and statistical analysis oflength distribution of reconstituted microtubules demonstrate that the increase inmean length of microtubules during each incubation period is in good accordancewith the increment in specific viscosity of the tubulin solution. These results withdark-field microscopy were further confirmed by observation of stained micro-tubules under a phase-contrast microscope. This indicates that increase in viscosityof the tubulin solution primarily reflects increase in microtubule length.

The effect of glycerol on tubulin or microtubules is well known. This reagent notonly maintains the polymerizability of the otherwise labile tubulin but also stabilizesreconstituted microtubules. In the present work, the stabilizing effect of glycerol wasreconfirmed quantitatively by analysing the number and length of microtubulesremaining after dilution of glycerol reassembly buffer. Dilution with reassembly buffercontaining no glycerol had a clear tendency to induce depolymerization of the micro-tubules, probably because the equilibrium between the polymeric and dimeric formsof tubulin was shifted towards the dimeric state. On the other hand, the presence ofglycerol reduces mean and maximal lengths of microtubules reconstituted. Thereason is not clear at the present time.

A demonstration of heterogeneous microtubule formation induced by mixing outerfibre fragments and tubulin dimers has been carried out by viscometric measurement(Kuriyama, 1975), and by observation with electron microscopy. The same processcould also be observed with light microscopy. Although the fine structure of theheterogeneous microtubules was not clear, the polarity and length of the reconstitutedmicrotubules could be determined. Allen & Borisy (1974) have presented detailedstudies on heterogeneous microtubules reconstituted from brain tubulin and flagellaraxonemes or outer doublets of Chlamydomonas. They indicated that a limited extentof growth on the B-tubule also occurred as well as proximal addition of heterogeneoustubulin.

The tubulin dimer fraction purified from partially purified tubulin by DEAE-Sephadex column chromatography could be substituted for molecular-sieved tubulindimers. When the fraction was chromatographed on a DEAE-Sephadex A-50column, 2 peaks appeared: one was a small peak eluted below 0-3 M KC1, which

614 R- Kuriyama and T. Miki-Noumura

contained little tubulin, and the other was a main fraction eluted with 0-5 M KC1,which contained tubulin alone, as determined by sodium dodecylsulphate/poly-acrylamide gel electrophoresis. As reported by Murphy & Borisy (1974), theseDEAE-purified tubulin dimers have a little ability to polymerize by themselves.However, a marked increase in viscosity could be induced by addition of the fractioneluted at 0-3 M KC1. This may indicate that nucleation or initiation of tubulinpolymerization in brain extract requires a protein component other than the micro-tubule fragment.

By electron-microscopic observation, it was possible to distinguish the reconstitutedmicrotubules from the outer fibre fragments used as nuclei, without any use ofDEAE-dextran or isotope labelling. The problem concerning polarity of polymerizingheterogeneous microtubule has been reported by Borisy et al. (1974) and Dentler,Granett, Witman & Rosenbaum (1974). Using decorated flagellar fragments orisotope-labelled microtubules as nuclei, they clearly demonstrated the unidirectionalgrowth of the microtubules. Furthermore, Borisy et al. (1974) reported that micro-tubules polymerized bidirectionally when purified tubulin of a higher protein concen-tration was used as the source of dimers. In the present work, the same result wasreproducibly observed: that microtubules are reconstituted from both ends of outerfibres, as shown in Figs. 6 and 7, at a protein concentration of 2 mg/ml. The lengthsof these bidirectionally reconstituted microtubules were usually unequal, whichsuggests that the 2 ends of the outer fibre fragments have unequal ability to incor-porate tubulin dimers.

Since observations with dark-field contrast permit the investigation of unfixedmicrotubules under more physiological conditions than can be used with othersystems, it is hoped that the use of this method will bring more and varied informationto advance our understanding of these structures.

The authors are greatly indebted to Prof. H. Sakai of the University of Tokyo and Prof. S.Asakura of Nagoya University for their valuable suggestions and discussions. Thanks areespecially due to Ms S. Endo for her kind aid with the electron microscopy and Dr J. C. Danfor her kind criticism in preparing the manuscript.

REFERENCES

ALLEN, C. & BORISY, G. G. (1974). Structural polarity and directional growth of microtubulesof Chlamydomonas flagella. J. molec. Biol. 90, 381-402.

BORISY, G. G., OLMSTED, J. B., MARCUM, J. M. & ALLEN, C. (1974). Microtubule assemblyin vitro. Fedn Proc. Fedn Am. Socs exp. Biol. 33, 167-174.

DENTLER, W. L., GRANETT, S., WITMAN, G. B. & ROSENBAUM, J. L. (1974). Directionalityof brain microtubule assembly in vitro. Proc. natn. Acad. Sci. U.S.A. 71, 1710-1714.

GASKIN, F., CANTOR, C. R. & SHELANSKI, M. L. (1974). Turbidimetric studies of the in vitroassembly and disassembly of porcine neurotubules. J. molec. Biol. 89, 737-758.

GIBBONS, I. R. (1965). Chemical dissection of cilia. Arcits Biol., Liege 76, 317-352.HAGA, T., ABE, T . & KUROKAWA, M. (1974). Polymerization and depolymerization of micro-

tubules in vitro as studied by flow birefringence. FEBS Letters, Amsterdam 39, 291-295.HASCHKE, R. H., BYERS, M. R. & FINK, B. R. (1974). Effects of lidocaine on rabbit brain

microtubular protein. J. Neurochem. 22, 837-843.HOTANI, H. (1974). A. Rep. Biophys. Soc. Japan 13, 303.


HOUSTON, L. L., ODELL, J., LEE, Y. C. & HIMES, R. H. (1974). Solvent isotope effects onmicrotubule polymerization and depolymerization. J. molec. Biol. 87, 141-146.

IINO, T., SUZUKI, H. & YAMAGUCHI, S. (1972). Reconstitution of Salmonella flagella attachedto cell bodies. Nature, New Biol. 237, 238-240.

IKAGAKUKENKYUJO GAKUYUKAI (1958). In Saikingaku Jissu Teiyo, p. 128. Tokyo: Maruzen.KAMIYA, R. & ASAKURA, S. (1974). A. Rep. Biophys. Soc. Japan 13, 304.KURIYAMA, R. (1975). Further studies on tubulin polymerization in vitro. J. Biochem., Tokyo

77,23-31-KURIYAMA, R. & SAKAI, H. (1974). Viscometric demonstration of tubulin polymerization.

J. Biocliem., Tokyo 75, 463-471.LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement

with the Folin phenol reagent. J. biol. Chem. 193, 265-275.MACNAB, R. & KOSHLAND, D. E. JR. (1974). Bacterial motility and chemotaxis: light-induced

tumbling response and visualization of individual flagella. J. molec. Biol. 84, 399-406.MURPHY, D. B. & BORISY, G. G. (1974). The role of tubulin-associated protein in microtubule

assembly. J. Cell Biol. 63, 236a.OLMSTED, J. B. & BORISY, G. G. (1973). Characterization of microtubule assembly in porcine

brain extract by viscometry. Biochemistry, N. Y. 12, 4282-4289.SHELANSKI, M. L., GASKIN, F. & CANTOR, C. R. (1973). Microtubule assembly in the absence

of added nucleotides. Proc. natn. Acad. Sci. U.S.A. 70, 765-768.STEPHENS, R. E. & LEVINE, E. E. (1970). Some enzymatic properties of axonemes from the

cilia of Pecten irradians. J. Cell Biol. 46, 416-421.SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine triphosphate-induced sliding of tubules

in trypsin-treated flagella of sea urchin sperm. Proc. natn. Acad. Sci. U.S.A. 68, 3092-3096.WEISENBERG, R. C. (1972). Microtubule formation in vitro in solutions containing low calcium

concentrations. Science, N.Y. 177, 1104-1105.WEISENBERG, R. C, BORISY, G. G. & TAYLOR, E. W. (1968). The colchicine-binding protein of

mammalian brain and its relation to microtubules. Biochemistry, N. Y. 7, 4466—4479.ZOBEL, C. R. (1973). Effect of solution composition and proteolysis on the conformation of

axonemal compositions. J. Cell Biol. 59, 573-594.{Received 19 March 1975)

6i6 R. Kuriyama and T. Miki-Noumura

Fig. 4. Dark-field micrographs of microtubules reconstituted from partially purifiedtubulin. 2-6 mg/ml tubulin fraction was incubated at 35 °C in glycerol-free reassemblybuffer at zero time, A, B, C, D and E are at o, 5, 10, 20 and 30 min, respectively. Scaleline represents 10/tm on all figures.

5A

Reconstituted microtubuies from brain tubulin 617

*

B V.

D

<

i }

\

• x ^ ^

. >

Fig. 5. Phase-contrast micrographs of microtubuies reconstituted from partiallypurified tubulin. A, B, c, D and E are at o, 5, 10, 20 and 30 min, respectively. Scaleline represents io/im on all figures.

40 C E L 19


Fig. 6. Heterogeneous microtubules observed under the light microscope, roml oftubulin dimer fraction (2 mg/ml), purified by gel filtration of partially purifiedtubulin through a Sephadex G-200 column, was mixed with 005 ml of a ciliary outerfibre fraction (17 mg/ml) and incubated at 35 °C for several hours. The scale linerepresents 10 fim on all figures, A-D, observed with dark-field contrast microscope.E-H, with the phase-contrast microscope.


>*"">•;

H '

620

7A

R. Kuriyama and T. Mitti-Noumura

B . C

I

0-1 //m

Fig. 7. Heterogeneous microtubules observed under the electron microscope afternegative staining with 1 % uranyl acetate. For conditions, see legend to Fig. 6. A showsa higher magnification of c, the area outlined, E shows the outer fibre fraction alone.

light-microscopic observations of individual microtubules … · 2017-06-25 · reconstituted...

Documents