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Synthesis and assembly of microtubule proteins in Tetrahymena pyriformis
Experimental
SYNTHESIS
AND
Cell Research 68 (I 97 1) I SO- I85
ASSEMBLY
OF MICROTUBULE
TETRAHYMENA
PROTEINS
IN
PYRIFORMIS
S. TAMURA Dcpartmenf
of Patholog.v, Nationrrl Institute of Health, Shinagan~a-ku, Tokyo, Japan
SUMMARY 1. Amounts of cilia-type microtubule proteins in a cell and a macronucleus were estimated to be about 7 1’0and 0.2 o,) of those of the total protein within the cell respectivelv. at various stages of the cell. These values imply that various microtubules in the cell in&de a common proteinls). 2. About 70 o,) of newly synthesized microtubule proteins were transferred to insoluble fraction within 3 min. This rapid transfer was proved to occur by the assembly of newly synthesized protein into insoluble structures, from the experimental results using cycloheximide and colchitine. 3. The synthetic activity of microtubule proteins and the rate of assembly of the proteins into higher structures (insoluble fration) were shown to be nearly constant through the stages after the end of the heat-treatment up to synchronous division.
It was previously reported that anti-ciliary microtubule serum can react with the proteins of isolated oral apparatus and of isolated macronuclei [3]. This implies that all microtubules present within Tetrahymena cell may include common protein subunit(s). If that is the case, it is surmised that Tetrahymena cells would have a considerably large amount of the proteins which react with anti-ciliary microtubule serum, as compared with the amount of ciliary microtubules in the cell. Furthermore, it is interesting to know when and how the microtubule proteins are synthesized and what changes occur in the existing state of the microtubule proteins, in accordance with the known events during cell cycle; e.g., development of a new oral apparatus, formation and disappearance of macronuclear microtubules involved in nuclear division, etc. [I, 2, 41. In the present studies, attempts were made to estimate the amount of cilia-type microExptl Cell Res 68
tubule proteins in the cell and to investigate the modes of synthesis and of assembly of the proteins with the cell life cycle.
MATERIAL
AND METHODS
Tctrahymena pyriformis W was used as a material. Culture conditions, isolation procedure of macronuclei and nrenaration of anti-ciliarv microtubule serum are the same as those of the previous paper, unless otherwise specified [3]. Details on specificity of anti-ciliary microtubule serum were also given.
Specific precipitin antigen
assay with radioactitre
Each 4 ml culture (about 5 x loj cells/ml) containing “C-amino acid mixture (0.2-I ,rKi/ml) was taken from selected stages. The labelled cells were washed with the inorganic solution and frozen with dry-ice acetone, followed by thawing at 26°C. They were transferred to a Teflon homogenizer with about 9 vol of cold distilled water and homogenized. Homogenate was dialysed against 10 mM Tris-HCI (pH 8.4) overnight in a cold condition and centrifuged at 10 000 g for 60 min at 4°C. The volume of supernatant was adjusted to 4.0 ml with Tris-HCI. The precipitate was dissolved in 0.1 N NaOH and further dialysed
Synthesis and assembly of microtubule proteins against 10 mM Tris-HCI (pH 8.4) overnight at 4°C. After dialysis, the volume of it was also adjusted to 4.0 ml with the buffer. Aliquots of the supernatant and the dissolved precipitate were kept aside to measure the total radioactivities and the protein amounts of each fraction. Another aliquot of each fraction was used for measuring the radioactivity of specific or non-specific precipitin resulting from antigenantibody reaction. To 0.5 ml of the supernatant or the dissolved precipitate was added each 0.5 ml of diluted anti-ciliary microtubule serum ( * 4, T 8, 16). After allowing the tubes with the mixture to stand for more than 24 h in a cold condition, 0.2 ml of non-radioactive microtubule proteins of cilia (1 mg/ml) were added to them as a carrier. The tubes were kept standing again for more than 24 h and centrifuged at 3 500 rpm for 60 min at 4°C. The supernatant was decanted and the precipitate was washed with IO mM Tris-HCI buffer (pH 8.4) by centrifugation. The precipitin was dried and dissolved in 0.2 ml of I N NaOH. Each 0.1 ml of the dissolved antigen-antibody complex was layed on 23 mm circular filter papers and dried. Radioactivities of the samples were measured with liquid-scintillation spectrometer (Beckman Model LS200B). As a control, rabbit antiserum against bovine serum albumin (BSA), having the same titre as anti-ciliary microtubule serum, and carrier BSA were always added to “C-labelled protein samples of Te~ahymena cells to detect non-specific radioactive precipitation. The vertitable counts of radioactive antigen reacted with anti-ciliary microtubule serum were thus obtained by deducting the counts derived from nonspecific contamination.
IS 1
RESULTS Amount of cilia-type microtubule fraction in the cell The amount of cilia-type microtubule fraction in the whole cell was estimated by the radioactivity of precipitate resulting from the reaction between antigen in homogenate of labelled cells and the antiserum to microtubule fraction of cilia. In this case, the cells were labelled with 14C-amino acid mixture for enough time (for about four generations) to regard labelled proteins as whole proteins of the cell. Tn these assay conditions, the counts derived from non-specific radioactive precipitation obtained by making use of anti-BSA serum and carrier BSA were about 1.5 O; of counts of each samples. As seen in table 1, the amounts of the microtubule proteins were estimated to be about 7 o,, of the total protein of the cell regardless of the phase of the cell. About 60 to 70 00 of the total microtubule
Table 1. Amounts of cilia-type microtubule proteins of ceN or macronucleus. The cells at three stages were long-term-labeled with 0.2 ,uCi/ml of 14C-amino acid mixture (for 17-18 h) Cell
Macronucleus (M. N.)
Total CPM
Microt. CPM
77095 68 544
4480 6798
I45 639
11 278
Microt./ Total “0
Total CPM
Microt. CPM
3.1 4.7 7.7
24670 3 980 28 670
591 310
2.0
901
3.1
2.2 4.9 7.1
IO 234
275
2.3
2.4 5.0 7.4
Microt./ Total “;,
Microt./ M. N. Prot./ Cell Cell Prot. “0
Log-phase S
P T
I.1 0.057
0.177
10 min after EHT S
P T
30949 28953 59902
1331 2923 4254
158
1.3
11 860
433
3.6
0.043
0.155
11493 1517
288
13010
418
2.2 I.0 3.2
0.030
0.096
1 626
70 min after EHT S
P T
30364 24533 54897
I 299 2762 4061
130
M. N., macronucleus. Microt., microtubule proteins. Prot., proteins. EHT, the end of the heat-treatment for synchronization. S, soluble fraction after centrifuging the cell homogenate at IO000 g for 60 min. P. the precipitated fraction. T, sum total of S and P fractions. Exprl
Cell Res 68
182 S. Tanwa
t-7 C-P
fraction were present in the centrifuge pellet which contained skeletal structures including cilia, kinetosomes, inter-kinetosomal fibers, oral apparatus and so on, and the rest of it was in the supernatant probably containing soluble microtubule proteins and/or dissociated fragments of labile microtubules in the cell. The ratio of the amount of the microtubule proteins in the soluble fraction to that in the insoluble fraction did not change so much whether the cells were taken from logphase or from two different stages of synchronized culture. On the other hand. amount of the microtubule fraction of a macronucleus was estimated to be about O.lLO.2 (I,, of total protein of the cell when homogenate of isolated macronuclei was used as a material containing antigen. Since little change in amount is found with the phase of the cell, the microtubule proteins seem to be always present in a macronucleus regardless of the stages of whether microtubules in the macronucleus are observed electron microscopically or not. Synthesis of cilia-type microtubule proteins and its transfer to insoluble structures
Fig. 1. Abscissa: min; ordinate: a, b, cpm; c, “,,. The synthesis of microtubule proteins and assembly of them into higher structures, with or without cycloheximide (IO {(g/ml). (a) Cumulative curves of “Camino acid incorporation into log-phase cells with or without cycloheximide. Abscissa represents lnin after labelling with 2 /Xi/ml of l&C-amino acid mixture. a-s, supernatant (or soluble) fraction of cell homogenate. u-p, precipitate (or insoluble) fraction of homogenate. O-s, supernatant fraction of cycloheximidetreated cells. h-p, precipitate fraction of cycloheximidetreated cells. (b) Cumulative curves of W-amino acid incorporation into ciliatype microtubule proteins with or without cycloheximide. a-p, microtubule proteins in precipitate. (I-S, microtubule proteins in supernatant. h-p, microtubule proteins in precipitate of cycloheximide-treated cells. h-s, microtubule proteins in supernatant of cycloheximide-treated cells. (c) Changes in percentages of soluble and inE.xptl Cell Rex 68
To investigate the modes of synthesis and of assembly of microtubule precursor proteins into higher structures, relatively short-term labelling with ‘“C-amino acid mixture was conducted. The curves a-s and a-p in fig. 1a show the cumulative increases of labelled proteins in the supernatant and the precipitate fraction of the cell, respectively. It is seen that proteins of the supernatant are synthesized at higher rate than that of the precipitate for first I.5 min, and only about
soluble fractions of cilia-type microtubule proteins with or without cycloheximide. Total cpm of each sample are taken as 100 and individual readings of supernatant or precipitate fraction are shown in percentages of the total. Recalculated from the result of (h). Other features are the same as in (h).
Sq.nthesis and assembly of microtubule proteins
183
Table 2. Suppression of transfer of newly synthesized microtubule proteins into structures b.v colchicine. Ten-min labeled cells nsith 2 pCi/ml of 14C-amino acid mixture Ir’ere chased for 30 min n.ith or without 30; colchicine. Increases in count after chasing are sh0lc.n in B-A and C-A to know the mode of transfer IO min-pulse label Microt. CPM (A) Supern. Precip. Microt.,
651 I 020 microtubule
fraction.
30 min-chase without Colchicine
30 min-chase with Colchicine
Microt. CPM (B)
B-A
Microt. CPM (C)
C-A
I 040 1 590
389 570
I 069 I I04
418 84
Super., supernatant
fraction
10 (‘,, of labelled proteins of the whole cell are prosent in the precipitate at 1.5 min after labelling. After 3 min, both proteins of the supernatant and of the precipitate are labelled at similar constant rate. On the other hand, as shown in fig. 1h, curves of increase of labelled microtubule proteins in the supernatant and the precipitate do not correspond to those of radioactive proteins in respective fractions: that is, incorporation of 14C-anlino acid mixture into microtubule proteins of the supernatant is almost the same as that of the precipitate for first 1.5 min. but in the subsequent time, radioactivities of the soluble microtubule fraction bccomc lower than those of the insoluble one which is always labelled at constant rate. For the sake of convenience of illustration, total cpm of each sampling point in fig. 1b are taken as 100 and the reading of soluble and insoluble fractions are shown in percentages of the total (fig. 1c). It is demonstrated from the curves a-p and a-s of fig. 1 c that about 70 oo of microtubule proteins are found in insoluble structures after 3 min; thereafter the values do not change so much. Under the presence of cycloheximide, both total protein synthesis and cilia-type microtubule protein synthesis are suppressed considerably (fig. 1a, 6, curves b-s and b-p). However, when the reading of soluble and
of the cell homogenate.
Precip., precipitate
fraction.
insoluble fractions are shown in percentages of the total as stated above, it is revealed that newly synthesized microtubule proteins are transferred to insoluble form pronouncedly as shown in fig. 1c (curves b-s and h-p) in comparison with those of normal conditions (curves a-s and a-p). This pronounced convertion may reflect the fact that under the cycloheximide inhibition, the amount of precursor protein is lessened by blockade of protein synthesis. while assembly of precursor proteins into microtubules may occur as usual. On the other hand, further experiments were made under such conditions as assembly of microtubule precursor proteins into higher structures fails to occur. with keeping the synthetic activity of the microtubule proteins as possible; namely, by chasing for 30 min under the presence of 3 “0 colchicine after 10 min-pulse labelling. As shown in columns B-A and C-A of table 2, which show the modes of transfer by increases in counts after chasing with colchicine or without respectively, the counts of labelled microtubule proteins in the supernatant increase while those in the precipitate decrease strikingly under the presence of colchicine as compared with the control. This suggests that transferance of newly synthesized microtubule proteins to insoluble structures is Esptl
Cdl
Res 68
184 S. Tamura very rapidly into insoluble structures in the normal condition as shown in fig. 1c (thin solid lines at the left side of the figure). Pulse-chase experiments in temperatureinduced synchronous culture were carried out to make sure whether or not synthetic activity of cilia-type microtubule proteins and the mode of assembly of them into higher structures change at each stage in the preparative process of division. At 10 min, 30 min and 50 min after the end of the heattreatment (EHT), l&C-amino acid mixture were added to synchronized culture for 5 min each and chased thereafter. Fig. 2a 6:~~z~~~~=~-oo o~~~~~A-0 shows the results of the pulseechase experiment. From the curve representing the change of the total amount of pulse-labelled micro50 tubule proteins it is seen that synthetic activities of the microtubule proteins are approximately the same at three stages after .-- ---T.-!~ -~-r+.-*: --. mom _e-g* EHT. The amount of this newly synthesized microtubule proteins corresponds to about ~.-. L.-I 0 IO 20 30 40 50 60 10 80 40 100 11’B120 IO:; of total labelled proteins of the cell Fig. 2. Abscissa: min after EHT; ordinate: 0, cpm; 0, :;. at each stage. Furthermore, the amounts of (a) Synthetic activities and natures of cilia-type pulse-labelled microtubule proteins in the microtubule proteins in pulse-chase experiments at three stages in the process of synchronous division. soluble and the insoluble fractions at each Synchronized cells were exposed to 14C-annno acid stage are about 20% and 80% of the total mixture for 5 min in the three periods: lo-15 min, 30-35 min, 50-55 min after the end of the heat labelled microtubule proteins, respectively. treatment (EHT). After chasing, counts of the microBy chasing the values seem not to change so tubule proteins in soluble and insoluble fractions were estimated by precipitin assay with antigen-antibody much through the stages after EHT. This reaction. Supernatant fraction; closed marks. precican clearly bc seen when the radioactivities pitate fraction; open marks. Circles, triangles and squares represent counts from the samples chased of the soluble and the insoluble microtubule at 15, 35 and 55 min after EHT, respectively. Shaded fractions arc expressed in percentages of the area shows the period of synchronous division. Solid bars in abscissarepresent labelling-terms. 0 -A--B ; total as shown in fig. 2b. The results suggest curve of synthetic activity of total cilia-type microthat the bulk of newly synthesized microtubu!e proteins. (b) Changes in percentages of soluble and insoluble fractions of cilia-type microtubule tubule proteins is rapidly assembled into proteins in pulse-chase experiment during synchroninsoluble structures in the same manner at ous division. Recalculated from the result of (a). Other details are the same as in (a). any stages after EHT. loo-
b
suppressed by colchicine and as the result, soluble proteins accumulate in supernatant. From these inhibition experiments, it is possible to consider that most newly synthesized microtubular proteins are assembled Exvtl Cd Res 68
DISCUSSION The result that the amount of microtubule proteins, which can react with anti-ciliary microtubule serum, is about 7 ‘$6 of total
Synthesis and assemblJ1of microtubule proteins Am,no Ac,ds
---)
Soluble Precursor --) Prolelns
t C_yc~ohe;l~lde
Insoluble
,
Micoluble Structruclu,es
Cdch?c_lne
f?,q. 3. Schematical illustration of the formation process of insoluble microtubule-structures. Arrow with dotted line represents the blockage region by cycloheximide or colchicine.
protein of the cell, was obtained by assuming that almost all proteins in the cell were labelled in our experimental conditions. Since this value is much higher than that expected as microtubule proteins of cilia, about 1 ob of total protein of the cell (unpublished data), this may also support the view that microtubules in various organella other than cilia include proteins common to microtubule proteins of cilia. On the modes of synthesis of this ciliatype microtubule proteins and of assembly of the proteins into higher structures, the following results were obtained; that is, most of newly synthesized proteins (70-80 :a) were rapidly converted to insoluble forms within about 3 min. Under the suppression of the synthesis of precursor proteins with cycloheximide, newly synthesized microtubule proteins were transferred as usual to insoluble forms. On the other hand, by the inhibition of polymerization with colchicine, newly synthesized microtubule proteins failed to transfer to insoluble structures, being accumulated in supernatant. Therefore, in conditions, assembly of the newly synthesized microtubule proteins into higher structures appear to occur within a short time through the pathway shown in fig. 3. In Tetrahymena cells, development of a
185
new oral apparatus and formation of macronuclear microtubules and others occur in the preparative process of division. However, the experimental data showed that the synthetic activity of cilia-type microtubular prccursor proteins and the rate of conversion of the proteins to higher structures remained nearly constant with the selected three stages after EHT, including the prc-division stage (fig. 2a, b). The reason why these activities remained unchanged with stages are unknown, but this dots not mean that synthesis and utilization of the microtubule proteins have nothing to do with division. In fact, by the addition of cycloheximide or colchicine to synchronous culture at EHT, the cells are completely prevented from entering division [3]. It is, therefore, inferred that constant synthesis of the microtubule proteins and successive rapid transfer to insoluble structures would bc requisites for progression of the cell cycle. The author wishes to thank Dr Y. Watanabe for his constant guidance throughout this study. He is also grateful to Dr Y. Egashira, Chief of the Department, and to Professor K. Matsui, Department of Zoology, Tokyo University of Education, for their great interest- and help.
REFERENCES 1. Frankel, J, Compt rend trav lab Carlsberg 33 (1962) I. 2. Tamura, S, Tsuruhara, T & Watanabe, Y, Exptl cell res 55 (1969) 351. 3. Tamura, S, Exptl cell res 68 (1971) 169. 4. Williams, N E & Zeuthen, E, Compt rend trav lab Carlsberg 35 (1966). Received December 8, 1970 Revised version received March 29, 1971
Exptl Cdl Res 68
SYNTHESIS
AND
Cell Research 68 (I 97 1) I SO- I85
ASSEMBLY
OF MICROTUBULE
TETRAHYMENA
PROTEINS
IN
PYRIFORMIS
S. TAMURA Dcpartmenf
of Patholog.v, Nationrrl Institute of Health, Shinagan~a-ku, Tokyo, Japan
SUMMARY 1. Amounts of cilia-type microtubule proteins in a cell and a macronucleus were estimated to be about 7 1’0and 0.2 o,) of those of the total protein within the cell respectivelv. at various stages of the cell. These values imply that various microtubules in the cell in&de a common proteinls). 2. About 70 o,) of newly synthesized microtubule proteins were transferred to insoluble fraction within 3 min. This rapid transfer was proved to occur by the assembly of newly synthesized protein into insoluble structures, from the experimental results using cycloheximide and colchitine. 3. The synthetic activity of microtubule proteins and the rate of assembly of the proteins into higher structures (insoluble fration) were shown to be nearly constant through the stages after the end of the heat-treatment up to synchronous division.
It was previously reported that anti-ciliary microtubule serum can react with the proteins of isolated oral apparatus and of isolated macronuclei [3]. This implies that all microtubules present within Tetrahymena cell may include common protein subunit(s). If that is the case, it is surmised that Tetrahymena cells would have a considerably large amount of the proteins which react with anti-ciliary microtubule serum, as compared with the amount of ciliary microtubules in the cell. Furthermore, it is interesting to know when and how the microtubule proteins are synthesized and what changes occur in the existing state of the microtubule proteins, in accordance with the known events during cell cycle; e.g., development of a new oral apparatus, formation and disappearance of macronuclear microtubules involved in nuclear division, etc. [I, 2, 41. In the present studies, attempts were made to estimate the amount of cilia-type microExptl Cell Res 68
tubule proteins in the cell and to investigate the modes of synthesis and of assembly of the proteins with the cell life cycle.
MATERIAL
AND METHODS
Tctrahymena pyriformis W was used as a material. Culture conditions, isolation procedure of macronuclei and nrenaration of anti-ciliarv microtubule serum are the same as those of the previous paper, unless otherwise specified [3]. Details on specificity of anti-ciliary microtubule serum were also given.
Specific precipitin antigen
assay with radioactitre
Each 4 ml culture (about 5 x loj cells/ml) containing “C-amino acid mixture (0.2-I ,rKi/ml) was taken from selected stages. The labelled cells were washed with the inorganic solution and frozen with dry-ice acetone, followed by thawing at 26°C. They were transferred to a Teflon homogenizer with about 9 vol of cold distilled water and homogenized. Homogenate was dialysed against 10 mM Tris-HCI (pH 8.4) overnight in a cold condition and centrifuged at 10 000 g for 60 min at 4°C. The volume of supernatant was adjusted to 4.0 ml with Tris-HCI. The precipitate was dissolved in 0.1 N NaOH and further dialysed
Synthesis and assembly of microtubule proteins against 10 mM Tris-HCI (pH 8.4) overnight at 4°C. After dialysis, the volume of it was also adjusted to 4.0 ml with the buffer. Aliquots of the supernatant and the dissolved precipitate were kept aside to measure the total radioactivities and the protein amounts of each fraction. Another aliquot of each fraction was used for measuring the radioactivity of specific or non-specific precipitin resulting from antigenantibody reaction. To 0.5 ml of the supernatant or the dissolved precipitate was added each 0.5 ml of diluted anti-ciliary microtubule serum ( * 4, T 8, 16). After allowing the tubes with the mixture to stand for more than 24 h in a cold condition, 0.2 ml of non-radioactive microtubule proteins of cilia (1 mg/ml) were added to them as a carrier. The tubes were kept standing again for more than 24 h and centrifuged at 3 500 rpm for 60 min at 4°C. The supernatant was decanted and the precipitate was washed with IO mM Tris-HCI buffer (pH 8.4) by centrifugation. The precipitin was dried and dissolved in 0.2 ml of I N NaOH. Each 0.1 ml of the dissolved antigen-antibody complex was layed on 23 mm circular filter papers and dried. Radioactivities of the samples were measured with liquid-scintillation spectrometer (Beckman Model LS200B). As a control, rabbit antiserum against bovine serum albumin (BSA), having the same titre as anti-ciliary microtubule serum, and carrier BSA were always added to “C-labelled protein samples of Te~ahymena cells to detect non-specific radioactive precipitation. The vertitable counts of radioactive antigen reacted with anti-ciliary microtubule serum were thus obtained by deducting the counts derived from nonspecific contamination.
IS 1
RESULTS Amount of cilia-type microtubule fraction in the cell The amount of cilia-type microtubule fraction in the whole cell was estimated by the radioactivity of precipitate resulting from the reaction between antigen in homogenate of labelled cells and the antiserum to microtubule fraction of cilia. In this case, the cells were labelled with 14C-amino acid mixture for enough time (for about four generations) to regard labelled proteins as whole proteins of the cell. Tn these assay conditions, the counts derived from non-specific radioactive precipitation obtained by making use of anti-BSA serum and carrier BSA were about 1.5 O; of counts of each samples. As seen in table 1, the amounts of the microtubule proteins were estimated to be about 7 o,, of the total protein of the cell regardless of the phase of the cell. About 60 to 70 00 of the total microtubule
Table 1. Amounts of cilia-type microtubule proteins of ceN or macronucleus. The cells at three stages were long-term-labeled with 0.2 ,uCi/ml of 14C-amino acid mixture (for 17-18 h) Cell
Macronucleus (M. N.)
Total CPM
Microt. CPM
77095 68 544
4480 6798
I45 639
11 278
Microt./ Total “0
Total CPM
Microt. CPM
3.1 4.7 7.7
24670 3 980 28 670
591 310
2.0
901
3.1
2.2 4.9 7.1
IO 234
275
2.3
2.4 5.0 7.4
Microt./ Total “;,
Microt./ M. N. Prot./ Cell Cell Prot. “0
Log-phase S
P T
I.1 0.057
0.177
10 min after EHT S
P T
30949 28953 59902
1331 2923 4254
158
1.3
11 860
433
3.6
0.043
0.155
11493 1517
288
13010
418
2.2 I.0 3.2
0.030
0.096
1 626
70 min after EHT S
P T
30364 24533 54897
I 299 2762 4061
130
M. N., macronucleus. Microt., microtubule proteins. Prot., proteins. EHT, the end of the heat-treatment for synchronization. S, soluble fraction after centrifuging the cell homogenate at IO000 g for 60 min. P. the precipitated fraction. T, sum total of S and P fractions. Exprl
Cell Res 68
182 S. Tanwa
t-7 C-P
fraction were present in the centrifuge pellet which contained skeletal structures including cilia, kinetosomes, inter-kinetosomal fibers, oral apparatus and so on, and the rest of it was in the supernatant probably containing soluble microtubule proteins and/or dissociated fragments of labile microtubules in the cell. The ratio of the amount of the microtubule proteins in the soluble fraction to that in the insoluble fraction did not change so much whether the cells were taken from logphase or from two different stages of synchronized culture. On the other hand. amount of the microtubule fraction of a macronucleus was estimated to be about O.lLO.2 (I,, of total protein of the cell when homogenate of isolated macronuclei was used as a material containing antigen. Since little change in amount is found with the phase of the cell, the microtubule proteins seem to be always present in a macronucleus regardless of the stages of whether microtubules in the macronucleus are observed electron microscopically or not. Synthesis of cilia-type microtubule proteins and its transfer to insoluble structures
Fig. 1. Abscissa: min; ordinate: a, b, cpm; c, “,,. The synthesis of microtubule proteins and assembly of them into higher structures, with or without cycloheximide (IO {(g/ml). (a) Cumulative curves of “Camino acid incorporation into log-phase cells with or without cycloheximide. Abscissa represents lnin after labelling with 2 /Xi/ml of l&C-amino acid mixture. a-s, supernatant (or soluble) fraction of cell homogenate. u-p, precipitate (or insoluble) fraction of homogenate. O-s, supernatant fraction of cycloheximidetreated cells. h-p, precipitate fraction of cycloheximidetreated cells. (b) Cumulative curves of W-amino acid incorporation into ciliatype microtubule proteins with or without cycloheximide. a-p, microtubule proteins in precipitate. (I-S, microtubule proteins in supernatant. h-p, microtubule proteins in precipitate of cycloheximide-treated cells. h-s, microtubule proteins in supernatant of cycloheximide-treated cells. (c) Changes in percentages of soluble and inE.xptl Cell Rex 68
To investigate the modes of synthesis and of assembly of microtubule precursor proteins into higher structures, relatively short-term labelling with ‘“C-amino acid mixture was conducted. The curves a-s and a-p in fig. 1a show the cumulative increases of labelled proteins in the supernatant and the precipitate fraction of the cell, respectively. It is seen that proteins of the supernatant are synthesized at higher rate than that of the precipitate for first I.5 min, and only about
soluble fractions of cilia-type microtubule proteins with or without cycloheximide. Total cpm of each sample are taken as 100 and individual readings of supernatant or precipitate fraction are shown in percentages of the total. Recalculated from the result of (h). Other features are the same as in (h).
Sq.nthesis and assembly of microtubule proteins
183
Table 2. Suppression of transfer of newly synthesized microtubule proteins into structures b.v colchicine. Ten-min labeled cells nsith 2 pCi/ml of 14C-amino acid mixture Ir’ere chased for 30 min n.ith or without 30; colchicine. Increases in count after chasing are sh0lc.n in B-A and C-A to know the mode of transfer IO min-pulse label Microt. CPM (A) Supern. Precip. Microt.,
651 I 020 microtubule
fraction.
30 min-chase without Colchicine
30 min-chase with Colchicine
Microt. CPM (B)
B-A
Microt. CPM (C)
C-A
I 040 1 590
389 570
I 069 I I04
418 84
Super., supernatant
fraction
10 (‘,, of labelled proteins of the whole cell are prosent in the precipitate at 1.5 min after labelling. After 3 min, both proteins of the supernatant and of the precipitate are labelled at similar constant rate. On the other hand, as shown in fig. 1h, curves of increase of labelled microtubule proteins in the supernatant and the precipitate do not correspond to those of radioactive proteins in respective fractions: that is, incorporation of 14C-anlino acid mixture into microtubule proteins of the supernatant is almost the same as that of the precipitate for first 1.5 min. but in the subsequent time, radioactivities of the soluble microtubule fraction bccomc lower than those of the insoluble one which is always labelled at constant rate. For the sake of convenience of illustration, total cpm of each sampling point in fig. 1b are taken as 100 and the reading of soluble and insoluble fractions are shown in percentages of the total (fig. 1c). It is demonstrated from the curves a-p and a-s of fig. 1 c that about 70 oo of microtubule proteins are found in insoluble structures after 3 min; thereafter the values do not change so much. Under the presence of cycloheximide, both total protein synthesis and cilia-type microtubule protein synthesis are suppressed considerably (fig. 1a, 6, curves b-s and b-p). However, when the reading of soluble and
of the cell homogenate.
Precip., precipitate
fraction.
insoluble fractions are shown in percentages of the total as stated above, it is revealed that newly synthesized microtubule proteins are transferred to insoluble form pronouncedly as shown in fig. 1c (curves b-s and h-p) in comparison with those of normal conditions (curves a-s and a-p). This pronounced convertion may reflect the fact that under the cycloheximide inhibition, the amount of precursor protein is lessened by blockade of protein synthesis. while assembly of precursor proteins into microtubules may occur as usual. On the other hand, further experiments were made under such conditions as assembly of microtubule precursor proteins into higher structures fails to occur. with keeping the synthetic activity of the microtubule proteins as possible; namely, by chasing for 30 min under the presence of 3 “0 colchicine after 10 min-pulse labelling. As shown in columns B-A and C-A of table 2, which show the modes of transfer by increases in counts after chasing with colchicine or without respectively, the counts of labelled microtubule proteins in the supernatant increase while those in the precipitate decrease strikingly under the presence of colchicine as compared with the control. This suggests that transferance of newly synthesized microtubule proteins to insoluble structures is Esptl
Cdl
Res 68
184 S. Tamura very rapidly into insoluble structures in the normal condition as shown in fig. 1c (thin solid lines at the left side of the figure). Pulse-chase experiments in temperatureinduced synchronous culture were carried out to make sure whether or not synthetic activity of cilia-type microtubule proteins and the mode of assembly of them into higher structures change at each stage in the preparative process of division. At 10 min, 30 min and 50 min after the end of the heattreatment (EHT), l&C-amino acid mixture were added to synchronized culture for 5 min each and chased thereafter. Fig. 2a 6:~~z~~~~=~-oo o~~~~~A-0 shows the results of the pulseechase experiment. From the curve representing the change of the total amount of pulse-labelled micro50 tubule proteins it is seen that synthetic activities of the microtubule proteins are approximately the same at three stages after .-- ---T.-!~ -~-r+.-*: --. mom _e-g* EHT. The amount of this newly synthesized microtubule proteins corresponds to about ~.-. L.-I 0 IO 20 30 40 50 60 10 80 40 100 11’B120 IO:; of total labelled proteins of the cell Fig. 2. Abscissa: min after EHT; ordinate: 0, cpm; 0, :;. at each stage. Furthermore, the amounts of (a) Synthetic activities and natures of cilia-type pulse-labelled microtubule proteins in the microtubule proteins in pulse-chase experiments at three stages in the process of synchronous division. soluble and the insoluble fractions at each Synchronized cells were exposed to 14C-annno acid stage are about 20% and 80% of the total mixture for 5 min in the three periods: lo-15 min, 30-35 min, 50-55 min after the end of the heat labelled microtubule proteins, respectively. treatment (EHT). After chasing, counts of the microBy chasing the values seem not to change so tubule proteins in soluble and insoluble fractions were estimated by precipitin assay with antigen-antibody much through the stages after EHT. This reaction. Supernatant fraction; closed marks. precican clearly bc seen when the radioactivities pitate fraction; open marks. Circles, triangles and squares represent counts from the samples chased of the soluble and the insoluble microtubule at 15, 35 and 55 min after EHT, respectively. Shaded fractions arc expressed in percentages of the area shows the period of synchronous division. Solid bars in abscissarepresent labelling-terms. 0 -A--B ; total as shown in fig. 2b. The results suggest curve of synthetic activity of total cilia-type microthat the bulk of newly synthesized microtubu!e proteins. (b) Changes in percentages of soluble and insoluble fractions of cilia-type microtubule tubule proteins is rapidly assembled into proteins in pulse-chase experiment during synchroninsoluble structures in the same manner at ous division. Recalculated from the result of (a). Other details are the same as in (a). any stages after EHT. loo-
b
suppressed by colchicine and as the result, soluble proteins accumulate in supernatant. From these inhibition experiments, it is possible to consider that most newly synthesized microtubular proteins are assembled Exvtl Cd Res 68
DISCUSSION The result that the amount of microtubule proteins, which can react with anti-ciliary microtubule serum, is about 7 ‘$6 of total
Synthesis and assemblJ1of microtubule proteins Am,no Ac,ds
---)
Soluble Precursor --) Prolelns
t C_yc~ohe;l~lde
Insoluble
,
Micoluble Structruclu,es
Cdch?c_lne
f?,q. 3. Schematical illustration of the formation process of insoluble microtubule-structures. Arrow with dotted line represents the blockage region by cycloheximide or colchicine.
protein of the cell, was obtained by assuming that almost all proteins in the cell were labelled in our experimental conditions. Since this value is much higher than that expected as microtubule proteins of cilia, about 1 ob of total protein of the cell (unpublished data), this may also support the view that microtubules in various organella other than cilia include proteins common to microtubule proteins of cilia. On the modes of synthesis of this ciliatype microtubule proteins and of assembly of the proteins into higher structures, the following results were obtained; that is, most of newly synthesized proteins (70-80 :a) were rapidly converted to insoluble forms within about 3 min. Under the suppression of the synthesis of precursor proteins with cycloheximide, newly synthesized microtubule proteins were transferred as usual to insoluble forms. On the other hand, by the inhibition of polymerization with colchicine, newly synthesized microtubule proteins failed to transfer to insoluble structures, being accumulated in supernatant. Therefore, in conditions, assembly of the newly synthesized microtubule proteins into higher structures appear to occur within a short time through the pathway shown in fig. 3. In Tetrahymena cells, development of a
185
new oral apparatus and formation of macronuclear microtubules and others occur in the preparative process of division. However, the experimental data showed that the synthetic activity of cilia-type microtubular prccursor proteins and the rate of conversion of the proteins to higher structures remained nearly constant with the selected three stages after EHT, including the prc-division stage (fig. 2a, b). The reason why these activities remained unchanged with stages are unknown, but this dots not mean that synthesis and utilization of the microtubule proteins have nothing to do with division. In fact, by the addition of cycloheximide or colchicine to synchronous culture at EHT, the cells are completely prevented from entering division [3]. It is, therefore, inferred that constant synthesis of the microtubule proteins and successive rapid transfer to insoluble structures would bc requisites for progression of the cell cycle. The author wishes to thank Dr Y. Watanabe for his constant guidance throughout this study. He is also grateful to Dr Y. Egashira, Chief of the Department, and to Professor K. Matsui, Department of Zoology, Tokyo University of Education, for their great interest- and help.
REFERENCES 1. Frankel, J, Compt rend trav lab Carlsberg 33 (1962) I. 2. Tamura, S, Tsuruhara, T & Watanabe, Y, Exptl cell res 55 (1969) 351. 3. Tamura, S, Exptl cell res 68 (1971) 169. 4. Williams, N E & Zeuthen, E, Compt rend trav lab Carlsberg 35 (1966). Received December 8, 1970 Revised version received March 29, 1971
Exptl Cdl Res 68