J. Cell Sci. 19, 607-620 (1975)
Printed in Great Britain
607
LIGHT-MICROSCOPIC OBSERVATIONS OF
INDIVIDUAL MICROTUBULES
RECONSTITUTED FROM BRAIN TUBULIN
RYOKO KURIYAMA AND TAIKO MIKI-NOUMURA
Department of Biophysics and Biochemistry, University of Tokyo,
and Institute of Molecular Biology, Nagoya University, Nagoya, Japan
SUMMARY
The course of polymerization of individual brain microtubules could be observed with a
light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity
change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed
with fixed and stained microtubules, using a phase-contrast microscope.
Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl
reagents such as £-chloromercuriphenyl sulphonic acid (PCMPS) and ^-chloromercuribenzoic
acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which
were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.
INTRODUCTION
The microtubules which are found in various kinds of organisms are supposed to
take part in cellular motility, transportation of cellular constituents or maintenance of
cell 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 depolymerization.
In 1972, Weisenberg (1972) demonstrated the reconstitution of brain microtubules
in vitro; this work has been confirmed by several groups of investigators. In all of
these studies, the process of microtubule assembly was analysed by electronmicroscopic observation and by physicochemical methods, such as measurement of
viscosity (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 native
state under a light microscope using dark-field illumination. Recently, Hotani (1974)
succeeded in visualizing individual flagella of Salmonella and photographing them by
using a light microscope of higher efficiency equipped with dark-field optics. Employing a similar optical system and technique for micrography, a reversible morphological
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R. Kuriyama and T. Miki-Noumura
transformation of the bacterial flagellum during pH changes was demonstrated by
Kamiya & Asakura (1974).
Although Summers & Gibbons (1971) have shown dark-field micrographs of
axonemes or of doublet microtubules from sperm tails, a singlet microtubule has not
yet been observed. Since its diameter (about 24 nm) is greater than that of the
Salmonella flagellum (15 nm), it was suggested that singlet microtubules ought to be
visible with dark-field illumination. This was first achieved with rabbit brain microtubules by Miki-Noumura and Kamiya (manuscript in preparation), who found that
the reconstituted singlet microtubule was quite straight.
In the present paper, we report observations of native microtubules reconstituted
from porcine brain tubulin, made with dark-field optics, and of silver-stained microtubules observed with a phase-contrast microscope. The process of tubulin polymerization was analysed by measurements on photographs of the average lengths of
reconstituted microtubules in comparison with the viscometric change during
polymerization. Besides the homogeneous microtubules of brain tubulin, the process
of assembly of heterogeneous microtubules derived from ciliary outer fibres of
Tetrahymena 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 purified
tubulin fraction (recovered after 1 cycle of polymerization and depolymerization), and a
tubulin dimer fraction (trailing fraction obtained by Sephadex G-200 gel nitration of the
partially purified tubulin fraction) were prepared as described elsewhere (Kuriyama & Sakai,
1974; Kuriyama, 1975). A purified tubulin dimer preparation was also obtained by linear
gradient elution of the partially purified fraction through a DEAE-Sephadex A-50 column or
by 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 min
MgSOa, 1 0 mM EGTA and i-o mM A T P (pH 6 7 ) . The reassembly buffer consisted of 50 mM
or 100 mM KC1, 0 5 mM MgSO 4 , i-o mM EGTA, i-o mM ATP, 5 mM MES (2-(iV-morpholino)ethanesulphonic acid)-KOH buffer (pH 6 7 ) .
Ciliary outer fibres were obtained from Tetrahymena pyriformis by the methods of Gibbons
(1965) and Stephens & Levine (1970), with some modifications. The axoneme fraction was
dialysed overnight against 200 vol. of 1 mM Tris-HCl, 0 1 mM EDTA, o-i mM dithiothreitol
at p H 8 o , followed by centrifugation at 40000 g for 30 min. The pellet was resuspended and
dialysed overnight against the same solution. After centrifugation, the outer fibre pellet was
suspended 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 type
microscope equipped with Olympus DC condenser (N.A. 1-20-1-33), an Olympus apochromatic objective lens (40 x , oil immersion type, N.A. i-o), a Nikon 10 x eye piece and a Nikon
E F M 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 Photo
Film Co.).
The silver nitrate method is a technique commonly employed to visualize bacterial flagella
under the light microscope (lino, Suzuki & Yamaguchi, 1972). Fixation and staining of microtubules were done following the method of Nishizawa & Sugawara (Ikagakukenkyujo
Gakuyukai, 1958), with some modifications. One drop of sample was put on a well cleaned
slide, which was then immersed in fixing solution ( 4 % tannic acid, o-oi % FeCl s , 5 %
glutaraldehyde, 0 0 0 8 % NaOH) for 10-15 rnin, thoroughly rinsed with distilled water, and
stained with 4 % silver nitrate in aqueous ammonia. After it became brownish, it was washed
again 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 were
postfixed 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 a
phase-contrast microscope, Nikon SUR-K type. Photographs were taken at a magnification of
1000 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 observations
were 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 of
tubulin was incubated at 35 °C in the reassembly buffer or in buffer containing
4 M glycerol, microtubules were reconstituted, causing an increase in viscosity as
described previously (Kuriyama & Sakai, 1974; Kuriyama, 1975). After the viscosity
reached a maximum value, the fraction was diluted with 100-200 vol. of reassembly
buffer containing 4 M glycerol (glycerol reassembly buffer) to permit clear observation
of individual microtubules. It was confirmed that addition of glycerol to the reassembly
buffer was the most effective way of preventing depolymerization of the reconstituted microtubules upon dilution.
Fig. 4 shows dark-field photographs of microtubules reconstituted from such a
partially purified tubulin fraction in glycerol-free reassembly buffer at intervals after
incubation at 35 °C. Observations on fixed and stained microtubules were carried
out using reconstituted microtubules diluted with glycerol reassembly buffer or with
buffer containing 2-5% glutaraldehyde. After being stained by the silver nitrate
method, the reconstituted microtubules were observed under a phase-contrast
microscope (Fig. 5). Each line observed in both figures is thought to correspond to
one singlet microtubule, since the diameter of ciliary outer doublet microtubules
observed with dark-field or phase-contrast microscopy was seen to be evidently wider
than the thickness of the lines in Figs. 4, 5 (data not shown). In order to make clearer
the point that each line was equivalent to an individual microtubule, the total number
and length of lines found in an aliquot of diluted sample were measured with both
the light microscope and the electron microscope. The results of a series of such
determinations indicated that the number and length distribution of lines obtained
with the light microscope agreed with those of microtubules counted and measured
with the electron microscope, within an error of 10-30 %. Therefore, the lines observed
light-microscopically can justifiably be identified as individual microtubules, and will
be called microtubules in this paper.
In the observations under the dark-field microscope, the polymerization process
could be followed on a slide by keeping the temperature of the mechanical stage
constant at 35 °C. Such unfixed microtubules depolymerized and disappeared
immediately when a solution of 2-7 mM ^-chloromercuriphenyl sulphonic acid
(PCMPS), 0-5 mM />-chloromercuribenzoic acid (PCMB) or 5 mM CaCl2 dissolved
in glycerol or glycerol-free reassembly buffer was added from one side of the cover-
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R. Kuriyama and T. Miki-Noumura
slip. Polymerized microtubules were also sensitive and responded readily to lower
temperature during observation. Details of these observations will be described in a
subsequent paper. On the other hand, the microtubules fixed with glutaraldehyde
showed no tendency to depolymerize even after immersion for longer periods in
reassembly buffer containing PCMPS, PCMB or CaCl2.
Correlation between length distribution of microtubules and viscosity increment during
polymerization
When partially purified tubulin was incubated at 35 °C, the viscosity of the solution
gradually increased with increase in the time of incubation (Fig. 1 A). In parallel with
the viscometric measurements, observations with dark-field illumination were carried
out. At intervals after the beginning of incubation, aliquots of the tubulin fraction
a- 0-6 -
10
15
20
25
Incubation time, mm
30
Fig. 1. A, viscosity increase in the course of tubulin polymerization. o-6 ml of partially
purified tubulin fraction (2-6 mg/ml) was incubated at 35 °C in glycerol-free reassembly
buffer 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 reconstituted
microtubules measured on the dark-field contrast photographs of Fig. 4. c, mean
lengths, in /im, of reconstituted microtubules measured on the phase-contrast
photographs of Fig. 5.
Reconstituted microtubules from brain tubulin
611
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. 4
are summarized in Fig. 2. The viscosity change was in good agreement with the
histograms shown in Fig. 2; that is, the increase in viscosity was closely correlated
with the increase in average length of the reassembled microtubules (Fig. 1 B). After
the plateau level of viscosity was attained there was no appreciable change in the
length distribution.
60
40
20
120
80
40
60
40 "5
20 3
- B
^ 40
2 20
o
I 40 o
S
C
20
20
40 - D
40
20
20
0
12
24
36
48
Length, ftm
Fig. 2
60
72
0
12
24 36 48
Length, fim
60
72
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 at
5, 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 of
microtubules 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 of
the silver-stained microtubules. Photographs of the stained microtubules at each
incubation period are shown in Fig. 5. The length distribution of the stained materials
measured on these photographs is summarized in Fig. 3. The increase in average
length of the reassembled microtubules during incubation also agreed well with the
increase in viscosity change in this case (Fig. 1 c).
It should be mentioned, however, that the mean length of reconstituted microtubules and the pattern of the length distribution were different when different
tubulin preparations were used. Furthermore, storage of tubulin fraction significantly
affected the time course of tubulin polymerization and maximum length of reconstituted microtubules obtained subsequently.
In contrast to microtubules from the partially purified tubulin fraction in glycerolfree reassembly buffer, the mean length of microtubules reconstituted from the
612
R. Kuriyama and T. Miki-Noumura
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 was
incubated at 35 °C, longer microtubules of nearly 100/tm could be reconstituted
(Kuriyama & Sakai, 1974). The viscosity increase of the preparation reached a
plateau level within 10-20 min. The average length of reassembled microtubules from
the crude tubulin fraction increased in parallel with the viscosity increment, as
reported in the previous paper (Kuriyama & Sakai, 1974).
Observation of heterogeneous microtubules reconstituted from brain tubulin dimers and
ciliary outer fibres
Further experiments on heterogeneous microtubule reassembly were performed
employing the optical systems described above. Measurement of viscosity and
observation with electron microscopy suggested that the purified tubulin dimers are
unable to reassemble into microtubules by themselves, and that fragments of microtubule are required as nuclei to induce the polymerization of tubulin (Kuriyama, 1975).
Ciliary outer fibres were therefore used as nuclei in the present experiment. Although
the total length of the outer fibres from Tetrahymena cilia was about 5 /tm, fragments
less than 2-3 /tm in length were obtained by dialysing the axoneme fraction for a
longer period. Such a treatment allowed A- and B-tubules of the fragments to remain
almost intact, as shown in Fig. 7E. When purified tubulin dimers in glycerol reassembly buffer were incubated with such outer fibres at 35 °C, heterogeneous
microtubules were reconstituted from one or both ends of the outer fibres as shown
in Fig. 6 A, B, E and F, and Fig. 7A-C. The length of the heterogeneous microtubules
became greater in parallel with the incubation time at 35 °C. Because of side-by-side
association among the outer fibres in reassembly buffer, reconstituted microtubules
tended to form clusters or radiating structures as shown in Fig. 6 c, D, G and H, and
Fig. 7D and F.
Similar results were obtained with a tubulin dimer fraction in glycerol reassembly
buffer purified by DEAE-Sephadex A-50 column chromatography. The viscosity of
this fraction, which was confirmed to contain only tubulin dimer, showed a slight
tendency to increase. The addition of a small amount of partially purified tubulin or
ciliary outer fibre preparation induced a striking increase in viscosity. On chromatography, a fraction eluted at 0—0-3 M KC1 contained a main component of high
molecular weight, which also promoted tubulin polymerization (data not shown).
In all of these cases, numerous reconstituted microtubules were identified at the
light-microscopic level.
As demonstrated in Fig. 7, observation with electron microscopy revealed that
polymerization of brain tubulin took place only from the A-tubule of ciliary outer
fibres, which agrees with the report by Borisy, Olmsted, Marcum & Allen (1974).
The length of the reconstituted microtubules formed in each incubation period in
glycerol reassembly buffer was as follows: less than 1 /tm during 30 min, 5-6/tm
during 3 h. After overnight incubation, much longer microtubules were formed. At
a tubulin dimer concentration of 2 mg/ml, microtubules were reconstituted from both
Reconstituted microtubules from brain tubulin
613
ends of the outer fibre fragments. These microtubules reconstituted from both ends
of an outer fibre were usually of unequal lengths.
DISCUSSION
The visualization of bacterial flagella by the use of a high-intensity arc lamp and
efficient optical systems (Macnab & Koshland, 1974; Hotani, 1974; Kamiya &
Asakura, 1974), suggested the possibility of identifying individual microtubules in an
unfixed state at the light-microscopic level. This was done by Miki-Noumura and
Kamiya (in preparation), who found that reconstituted rabbit brain microtubules had
a straight form. The same is true of the porcine brain microtubules used in the present
work. On the other hand, partially trypsin-digested axonemes of sperm tails exhibited
a coiled form, having a diameter of about 3 /im, as reported by Zobel (1973).
The present observations using dark-field illumination and statistical analysis of
length distribution of reconstituted microtubules demonstrate that the increase in
mean length of microtubules during each incubation period is in good accordance
with the increment in specific viscosity of the tubulin solution. These results with
dark-field microscopy were further confirmed by observation of stained microtubules under a phase-contrast microscope. This indicates that increase in viscosity
of the tubulin solution primarily reflects increase in microtubule length.
The effect of glycerol on tubulin or microtubules is well known. This reagent not
only maintains the polymerizability of the otherwise labile tubulin but also stabilizes
reconstituted microtubules. In the present work, the stabilizing effect of glycerol was
reconfirmed quantitatively by analysing the number and length of microtubules
remaining after dilution of glycerol reassembly buffer. Dilution with reassembly buffer
containing no glycerol had a clear tendency to induce depolymerization of the microtubules, probably because the equilibrium between the polymeric and dimeric forms
of tubulin was shifted towards the dimeric state. On the other hand, the presence of
glycerol reduces mean and maximal lengths of microtubules reconstituted. The
reason is not clear at the present time.
A demonstration of heterogeneous microtubule formation induced by mixing outer
fibre fragments and tubulin dimers has been carried out by viscometric measurement
(Kuriyama, 1975), and by observation with electron microscopy. The same process
could also be observed with light microscopy. Although the fine structure of the
heterogeneous microtubules was not clear, the polarity and length of the reconstituted
microtubules could be determined. Allen & Borisy (1974) have presented detailed
studies on heterogeneous microtubules reconstituted from brain tubulin and flagellar
axonemes or outer doublets of Chlamydomonas. They indicated that a limited extent
of growth on the B-tubule also occurred as well as proximal addition of heterogeneous
tubulin.
The tubulin dimer fraction purified from partially purified tubulin by DEAESephadex column chromatography could be substituted for molecular-sieved tubulin
dimers. When the fraction was chromatographed on a DEAE-Sephadex A-50
column, 2 peaks appeared: one was a small peak eluted below 0-3 M KC1, which
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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/polyacrylamide gel electrophoresis. As reported by Murphy & Borisy (1974), these
DEAE-purified tubulin dimers have a little ability to polymerize by themselves.
However, a marked increase in viscosity could be induced by addition of the fraction
eluted at 0-3 M KC1. This may indicate that nucleation or initiation of tubulin
polymerization in brain extract requires a protein component other than the microtubule fragment.
By electron-microscopic observation, it was possible to distinguish the reconstituted
microtubules from the outer fibre fragments used as nuclei, without any use of
DEAE-dextran or isotope labelling. The problem concerning polarity of polymerizing
heterogeneous microtubule has been reported by Borisy et al. (1974) and Dentler,
Granett, Witman & Rosenbaum (1974). Using decorated flagellar fragments or
isotope-labelled microtubules as nuclei, they clearly demonstrated the unidirectional
growth of the microtubules. Furthermore, Borisy et al. (1974) reported that microtubules polymerized bidirectionally when purified tubulin of a higher protein concentration was used as the source of dimers. In the present work, the same result was
reproducibly observed: that microtubules are reconstituted from both ends of outer
fibres, as shown in Figs. 6 and 7, at a protein concentration of 2 mg/ml. The lengths
of these bidirectionally reconstituted microtubules were usually unequal, which
suggests that the 2 ends of the outer fibre fragments have unequal ability to incorporate tubulin dimers.
Since observations with dark-field contrast permit the investigation of unfixed
microtubules under more physiological conditions than can be used with other
systems, it is hoped that the use of this method will bring more and varied information
to 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 are
especially due to Ms S. Endo for her kind aid with the electron microscopy and Dr J. C. Dan
for her kind criticism in preparing the manuscript.
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{Received 19 March 1975)
HOUSTON,
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R. Kuriyama and T. Miki-Noumura
Fig. 4. Dark-field micrographs of microtubules reconstituted from partially purified
tubulin. 2-6 mg/ml tubulin fraction was incubated at 35 °C in glycerol-free reassembly
buffer at zero time, A, B, C, D and E are at o, 5, 10, 20 and 30 min, respectively. Scale
line represents 10/tm on all figures.
Reconstituted microtubuies from brain tubulin
617
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Fig. 5. Phase-contrast micrographs of microtubuies reconstituted from partially
purified tubulin. A, B, c, D and E are at o, 5, 10, 20 and 30 min, respectively. Scale
line represents io/im on all figures.
40
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R. Kuriyama and T. Miki-Noumura
Fig. 6. Heterogeneous microtubules observed under the light microscope, r o m l of
tubulin dimer fraction (2 mg/ml), purified by gel filtration of partially purified
tubulin through a Sephadex G-200 column, was mixed with 005 ml of a ciliary outer
fibre fraction (17 mg/ml) and incubated at 35 °C for several hours. The scale line
represents 10 fim on all figures, A-D, observed with dark-field contrast microscope.
E-H, with the phase-contrast microscope.
Reconstituted microtubules from brain tubulin
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620
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R. Kuriyama and T. Mitti-Noumura
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.
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Fig. 7. Heterogeneous microtubules observed under the electron microscope after
negative staining with 1 % uranyl acetate. For conditions, see legend to Fig. 6. A shows
a higher magnification of c, the area outlined, E shows the outer fibre fraction alone.