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A temperature-sensitive mutant defective in mitosis and cytokinesis
Pr~nrrd ,n Sweden CopvriRht 0 1976 by Academic Press. Inc. A// ri,qhrs of rrproducrion in anyform resewed
Experimental Cell Research 100 (1976) 297-302
A TEMPERATURE-SENSITIVE MITOSIS
MUTANT
DEFECTIVE
IN
AND CYTOKINESIS
T. SHIOMI and K. SAT0 Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University, Yamadakami, Suita 565, and Division of Genetics, National Institute of Radiological Sciences, Anagawa, Chiba 280, Japan
SUMMARY A temperature-sensitive mutant, ts2, of murine leukemic cells (L5178Y) loses its viability gradually at the non-permissive temperature (39°C) but resumes normal growth when shifted to the permissive temperature (33°C). At 39°C the incorporation rate of thymidine is reduced on a per-cell-basis, whereas that of uridine and leucine is unchanged. Autoradiographic study indicates that the fraction of cells which can synthesize DNA decreases steadily with time of incubation at 39°C. Accumulation of mitotic and multinucleate cells suggests that ts2 cells are defective in both mitosis and cytokinesis. Experiments using synchronized culture demonstrate that the cells shifted up at the G2, but not at the Gl phase pass through the first mitotic phase normally.
It is well known that life cycle of mammalian cells can be divided into four phases, Gl, S, G2, and M [l]. This ordered sequence is under genetic control and specific factors synthesized during the individual phases are supposed to allow the cell to enter the succeeding phases. If we can control the synthesis or activity of factors which are necessary for cell cycle traverse, it will be of great help to understand the mechanisms regulating the cell cycle. In this respect, it seems useful to isolate temperature-sensitive mutants which are blocked in a certain phase of the cell cycle. By now several cell cycle mutants have been reported [2-71. We have isolated ten temperature-sensitive mutants of L5178Y murine leukemic cells. Two of these mutants, tsl and ts3, have been characterized [8,9]. In this paper we describe a mutant, ts2, 20-761805
whose mitosis and cytokinesis are affected at the non-permissive temperature. An understanding of the properties of this mutant may throw some light on the mechanisms involved in the progression of cell division. MATERIALS
AND METHODS
Cell culture conditions L5178Y cells were grown in enriched Eagle’s MEM medium (Eagle’s MEM plus 13 pg/ml of L-asparagine, 13 &g/ml of L-aspartic acid, 9 pg/ml of L-alanine, 11 &ml -. of L-serine, 35 &ml of L-oroline, 15 &ml of L-glutamic acid, 8 p&ml of gly&e and 9 &/ml of folic acid) supplemented with 10% V/V calf serum. Cell numbers were determined by counting in a hemocytometer or in a Coulter counter, model ZB. Soft agar cloning technique was previously described [8].
Isolation of mutant 02 L5 178Y cells were treated with 0.1 pg/ml N-methylN’-nitro-N-nitrosoguanidine for 2 h which gave a survival fraction of 21%. The cells were washed and incubated in fresh medium for further 48 h at 33°C. The culture was shifted up to 39°C and after 16 h, 0.2 pg/ml cytosine arabinoside was added. After 24 h the cells Exp
Cell
Res
100 (1976)
298
Shiomi and Sato
lob-
fixed with methanol followed by Giemsa staining. For autoradiography the cells, pulse-labeled for I h with 0.5 &i/ml of r3H]TdR (spec. act. 14.5 Cilmmole), were smeared on coverslips, fixed with methanol and treated with 2 % ice-cold perchloric acid for 40 min in order to wash out the acid-soluble [YH]TdR. The coverslips were washed twice with water, dried and then dipped in nuclear track emulsion, NR-M2, Sakura. After appropriate time of exposure (5 to 7 days), the coverslips were developed and stained with Giemsa prior to counting.
Synchronization
0
2
4
6
R
time (days); ordinate: cell no. Growth curves of wild type (O-O, 0-O) and ts2 (O-0, U-m) cells after temperature shift from 39°C (solid symbols) to 33°C (open symbols). The arrows indicate when shift-down was made.
Synchronized cell population was prepared according to the method described by Doida & Okada [IO]. In order to apply their method to our culture conditions, the following modifications were made. Exponentially growing cells were incubated with lO-3 M of thymidine for 8 h at 33°C. Then they were collected by centrifugation and resuspended in the prewarmed fresh medium containing IOe6 M of deoxycytidine. After 5 h, colcemid was added at a final concentration of 0.05 pg/mI and incubated for 5 h. After re-
Fig. I. Abscissa:
were washed, plated in soft agar medium and incubated at 33°C. The developed colonies were tested for growth at 33°C and 39°C. A mutant, ts2, was used for the present study.
2 a 1
0.5 b
Measurement of macromolecular synthesis All the radiochemicals were purchased from Daiichi Pure Chemicals Co., Ltd., Tokyo. Cells were pulselabeled for I h with I &i/ml of [3H]TdR (spec. act. 14.5 Ci/mmole), 1 &i/ml of [3H]uridine (15.0 Ci/m mole), or 0.5 pCi/ml of [Y-Llleucine (204.0 mCi/ mmole). When [3H]uridine was used, unlabeled thymidine and deoxycytidine were added together at IO pg/mI each to reduce the incorporation of uridine into DNA. To measure the incorporation of [Y-L]Ieucine into acid-insoluble fraction, media containing onetenth the normal concentration of leucine were used. Immediately after labeling, the cells were chilled and IOO-fold excess unlabeled precursors were added. They were then applied to the presoaked glass fiber filters (Whatman, type GF/F) and treated successively with phosphate buffered saline (PBS), ice-cold 5% trichloroacetic acid (TCA) and methanol, and the filters were counted as described before [8].
Light microscopic observation and autoradiography At appropriate times, cells were collected and washed twice with PBS. For light microscopic observation, the cells were smeared on slide glasses, dried and Exp Cell Rrs IO0 (1976)
t
0
10
20
30
time (hours); ordinate: rel. radioactivity per cell. Incorooration of (a) 13H1TdR: (b) 13Hluridine; (c) [‘4C-L]I&cine into wild-type (O-0, -0-O) and ts2 (O-0, m-m) cells at 33°C (open symbols) and 39°C (solid symbols). 2x I04~ceIIswere inoculated and cultured for 20 h at 33°C. Then half of cultures were switched to 39°C. After various lengths of time at 33°C and 39”C, radioactive precursors were added for 1 h and radioactivities incorporated into acidinsoluble materials were determined. At the same time cell numbers were counted in a Coulter counter and the incorporation was expressed on a per-cell-basis.
Fig. 2. Abscissa:
A division mutant of L5178Y
A
00'
doubling time of 17-19 h. At 39°C wild type cells grew well with a doubling time of 11 h, whereas ts2 cells ceased to grow after l-2 days and the cell number decreased thereafter. Note that when ts2 cells were cultured at a higher cell density (I x loj cells/ ml) at 39”C, their growth character was identical to the result shown in fig. 1. This property is different from that of the mutant reported by Wittes & Ozer [I 11, which showed density-dependent temperature sensitivity. The plating efficiencies in soft agar at 33°C and 39°C were 102% and 63 % for wild type cells, and 52% and 0.13 % for ts2 cells, respectively.
*
20
299
40
3. Abscissa: time (hours); ordinate: labeling index 6%). Changes in labeling index of wild type (O-O, 0-O) and ts2 (El-O, D-m) cells with time of incubation at 33°C (open symbols) and 39°C (solid symbols). 5x IO4cells were inoculated at time zero and the cultures were incubated at 33°C and 39°C. After various lengths of incubation, I h-pulse labeling of [3H]TdR was performed. The cells which had more than IO silver grains were counted as the cells synthesizing DNA.
Fig.
moving the drug by washing twice with PBS, the cells were used as a synchronized cell population. Under the experimental conditions, cells showed 50-70% synchrony and the duration of Gl, S, G2 and M was 3-4, 9-10, 34 and 0.5-l h, respectively.
RESULTS Cell growth at the permissive and non-permissive temperatures Cell growth of parental strain (wild type) and temperature-sensitive mutant ts2 was measured at the permissive (33°C) and nonpermissive (39°C) temperatures. As shown in tig. 1, at 33°C in liquid medium, wild type cells grew with a doubling time of 16-17 h and ts2 cells grew with a slightly longer
Temperature shift experiment Mutant cells kept at 39°C were shifted down to 33°C at various times and cell growth was measured. As seen in fig. 1, upon shiftdown cells resume growth with a doubling time comparable to that of control after delay of l-3 days, though the recovery of cell growth was retarded as the periods of heat exposure prolonged. This result shows that the mutant cells retain their ability for division even after maintenance at 39°C for as long as 4 days. Macromolecular synthesis at the non-permissive temperature In order to know the effect of temperature on ts2 cells, the synthesis of three types of macromolecules, DNA, RNA and proteins was examined. At various times, wild type and mutant cells (2-3~ lo4 cells/ml) were pulse-labeled with radioactive precursors for 1 h at either 33°C or 39°C. As shown in fig. 2a, at 39°C the incorporation rate of [3H]TdR on a per-cell-basis fell gradually in ts2 cells. However, uptake of [3H]uridine and [‘“Clleucine did not differ between 33°C and 39°C (fig. 2b, c).
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Shiomi and Sato
Fig. 4. Morphology of ts2 cells cultured at (a) 33°C; (b) 39°C for 20 h; and (c) 40 h. x 1300.
Labeling index The previous experiment (fig. 2~) showed that the incorporation rate of r3H]TdR into acid-insoluble material was affected at 39°C. In order to clarify this phenomenon further, the fraction of cells synthesizing DNA was measured autoradiographically. The appropriate number of cells were incubated at 33°C and half of them were switched to 39°C. At varying times, r3H]TdR was added to the cultures for 1 h and then samples were treated for autoradiography. As shown in fig. 3, the fraction of ts2 cells that were able to synthesize DNA remained constant at 33°C but decreased steadily at 39°C the labeling index of 68% at time zero being reduced to 41% after 36 h. In contrast, no difference was found in the labeling indices in wild type cells even at 36 h between 33°C and 39°C. Thus it is likely that the decrease in thymidine incorporation observed at 39°C in ts2 cells is due to the decreased fraction of cells synthesizing DNA. Morphological changes observed during incubation at 39°C When ts2 cells were grown at 33”C, normal mitotic figures were observed (fig. 4a), but Exp
Cell
Res
IW
(1976)
at 39°C many aberrant mitotic figures and multinucleate cells were found (fig. 4b). In the aberrant mitotic cells, chromosomes appear no longer to be held together by the spindles and are scattered in the cytoplasm. These figures resemble those whose spindle fiber formation is blocked by specific inhibitors such as colchicine. Many multi-
/
A
Fig. 5. Abscissa:
time (hours); ordinate: fraction of cells (%). Changes in mitotic index (O-O, 0-a) and percentage of multinucleate cells (A-A, A-A) in ts2 cells after various periods of incubation at 33°C (open symbols) and 39°C (solid symbols). I x 105cells were inoculated. The arrows indicate when shifts from 39°C to 33°C were made. The mitotic index is expressed as the percentage of cells engaged in mitosis. The cells which had more than one nucleus are scored as multinucleate cells.
A division mutant of L5178Y
301
The number of multinucleate cells increased with time of incubation as well. However, neither mitotic indices nor multinucleate cells changed in ts2 cells incubated at 33°C (fig. 5). When the cells incubated at 39°C were shifted to 33°C at 24 h, the mitotic cell fraction rather increased and the multinucleate cell fraction decreased slowly (fig. 5). Experiments with synchronized cells As can be seen in fig. 5, temperatureinduced mitotic arrest is not apparent until 6 h following shift-up. This result suggests that cells in the late G2 and M phases are not affected by the elevated temperature. Therefore, using the synchronously growing cells we examined which stage in a cell cycle was affected by temperature. Our synchronized population had the
I
I
0
20
I
40
Fig. 6. Abscissa: time (hours); ordinate: fraction of cells (%). (a) Change in mitotic index (O-O, 0-O); (b) percentage of binucleate cells (CL-O, H-W); and
(c) percentage of cells with more than two nuclei (A-A, A-A) in synchronized ts2 cells after various periods of incubation at 33°C (open symbols) and 39°C (solid symbols). After colcemid release cells in the Gl phase were shifted to 39°C (arrows). 0 10
nucleate cells were also found which have two or more normal sized nuclei, or two or more unequally separated lobes of a nucleus (fig. 4~). In order to determine the relation between aberrant mitosis and multinucleation, the number of cells showing these figures was scored at various times of incubation at the non-permissive temperature. As shown in fig. 5, in mutant cells, the mitotic indices began to increase at 6 h after shift to 39”C, reached the maximum of 24% at 20 h and decreased thereafter.
ii-i
0
20
40
time (hours); ordinate: fraction of cells (%). Symbols were the same as in fig. 6. Synchronized cells in the G2 phase were shifted to 39°C (arrows).
Fig. 7. Abscissa:
Exp Cd/Revs 100 (1976)
302
Shiomi and Sato
mitotic index of 50% at the time of colcemid release. Most of these cells passed through the mitotic phase after 3 h-incubation in the colcemid-free medium, traversed the cell cycle and synchronously entered the second mitotic phase after 14 h (figs 6a, 7a). The cells incubated at 33°C for 3 h and 13 h after release from colcemid block were used as the GI and G2 cell populations, respectively. When cells were shifted up at the Gl phase, the first peak of mitotic indices was much higher than that of the cells cultured at the permissive temperature (fig. 6a). As the mitotic indices decreased, binucleate cells accumulated quickly (fig. 6b) and the increase in the fraction of cells with more than two nuclei followed with some delay (fig. 6~). At 33°C binucleate cells did not accumulate at all. On the other hand, when cells in G2 phase were switched to the non-permissive temperature, the first cell division appeared to occur normally, but the second one was affected, namely, a much higher peak of mitotic indices and the accumulation of binucleate cells (fig. 7a, b). In synchronized wild type cells, no difference in the mitotic indices was observed between 33°C and 39°C. Thus upon shift-up cells in the Gl phase show abnormal mitosis and blocked cytokinesis, while the G2 cells can complete the first, but not the second division. DISCUSSION How do we account for the characteristic morphological changes and growth responses of ts2 cells? Mitotic arrest induced by the restrictive condition appears always to be followed by cytokinetic block in the present mutant. If this is the case, substances affected by temperature would be necessary for both mitosis and cytokinesis. Moreover, experiments with synchronized culture indicate that the temperature-sensiExp CellRcs lOO(l976)
tive stage in ts2 cells is located somewhere from the early Gl through the late G2 phase, though the temperature effect becomes apparent in the M phase. Three temperature-sensitive mutants with a defect in cytokinesis [4,6,7] and one mutant in mitosis [2] have been described. In contrast to these, the present mutant ts2 appears to be defective in both processes of cell division. Drugs such as colchicine and cytochalasin are known to block mitosis and cytokinesis, respectively. But no drugs have so far been reported which inhibit both processes. Considering these findings it is difficult to connect the defective function in ts2 cells with microtubules or microfilaments. Furthermore, the defectiveness is not manifested unless the elevated temperature is given far before the time of its expression. We are currently determining the more precise location of the heat-susceptible stage in the cell cycle in the mutant. The authors thank Drs A. Matsushiro and Y. Nishimune for discussions, Dr K. Mizobuchi for reading the manuscript, and Miss Naoko Hieda for assistance. This work was supported by grants-in-aid for Scientific and Cancer Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Howard, A & Pelt, S R, Heredity 6, suppl. (1953) 261. 2. Wane. R J. Nature 248 (1974) 76. 3. Liskay, R M, J cell physiol 8;1(1974)49. 4. Smith. B J & Wigglesworth, N M, J cell _physiol82 (1973)‘339. -5. Burstin, S J, Meiss, H K & Basilica, C, J cell physiol84 (1974) 397. 6. Thompson, L H & Baker, R M, Methods in cell biology (ed D M Prescott) vol. 6, p. 260. Academic Press, New York (1973). 7. Hatzfeld, J & Buttin, G, Cell 5 (1975) 123. 8. Sato, K & Shiomi, T, Exp cell res 88 (1974) 295. 9. Sato, K, Nature 257 (1975) 813. 10. Doida, Y & Okada, S, Exp cell res 48 (1967) 540. 11. Wittes, R E & Ozer, H L, Exp cell res 80 (1973) 127. Received January 30, 1976 Accepted February 5, 1976
Experimental Cell Research 100 (1976) 297-302
A TEMPERATURE-SENSITIVE MITOSIS
MUTANT
DEFECTIVE
IN
AND CYTOKINESIS
T. SHIOMI and K. SAT0 Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University, Yamadakami, Suita 565, and Division of Genetics, National Institute of Radiological Sciences, Anagawa, Chiba 280, Japan
SUMMARY A temperature-sensitive mutant, ts2, of murine leukemic cells (L5178Y) loses its viability gradually at the non-permissive temperature (39°C) but resumes normal growth when shifted to the permissive temperature (33°C). At 39°C the incorporation rate of thymidine is reduced on a per-cell-basis, whereas that of uridine and leucine is unchanged. Autoradiographic study indicates that the fraction of cells which can synthesize DNA decreases steadily with time of incubation at 39°C. Accumulation of mitotic and multinucleate cells suggests that ts2 cells are defective in both mitosis and cytokinesis. Experiments using synchronized culture demonstrate that the cells shifted up at the G2, but not at the Gl phase pass through the first mitotic phase normally.
It is well known that life cycle of mammalian cells can be divided into four phases, Gl, S, G2, and M [l]. This ordered sequence is under genetic control and specific factors synthesized during the individual phases are supposed to allow the cell to enter the succeeding phases. If we can control the synthesis or activity of factors which are necessary for cell cycle traverse, it will be of great help to understand the mechanisms regulating the cell cycle. In this respect, it seems useful to isolate temperature-sensitive mutants which are blocked in a certain phase of the cell cycle. By now several cell cycle mutants have been reported [2-71. We have isolated ten temperature-sensitive mutants of L5178Y murine leukemic cells. Two of these mutants, tsl and ts3, have been characterized [8,9]. In this paper we describe a mutant, ts2, 20-761805
whose mitosis and cytokinesis are affected at the non-permissive temperature. An understanding of the properties of this mutant may throw some light on the mechanisms involved in the progression of cell division. MATERIALS
AND METHODS
Cell culture conditions L5178Y cells were grown in enriched Eagle’s MEM medium (Eagle’s MEM plus 13 pg/ml of L-asparagine, 13 &g/ml of L-aspartic acid, 9 pg/ml of L-alanine, 11 &ml -. of L-serine, 35 &ml of L-oroline, 15 &ml of L-glutamic acid, 8 p&ml of gly&e and 9 &/ml of folic acid) supplemented with 10% V/V calf serum. Cell numbers were determined by counting in a hemocytometer or in a Coulter counter, model ZB. Soft agar cloning technique was previously described [8].
Isolation of mutant 02 L5 178Y cells were treated with 0.1 pg/ml N-methylN’-nitro-N-nitrosoguanidine for 2 h which gave a survival fraction of 21%. The cells were washed and incubated in fresh medium for further 48 h at 33°C. The culture was shifted up to 39°C and after 16 h, 0.2 pg/ml cytosine arabinoside was added. After 24 h the cells Exp
Cell
Res
100 (1976)
298
Shiomi and Sato
lob-
fixed with methanol followed by Giemsa staining. For autoradiography the cells, pulse-labeled for I h with 0.5 &i/ml of r3H]TdR (spec. act. 14.5 Cilmmole), were smeared on coverslips, fixed with methanol and treated with 2 % ice-cold perchloric acid for 40 min in order to wash out the acid-soluble [YH]TdR. The coverslips were washed twice with water, dried and then dipped in nuclear track emulsion, NR-M2, Sakura. After appropriate time of exposure (5 to 7 days), the coverslips were developed and stained with Giemsa prior to counting.
Synchronization
0
2
4
6
R
time (days); ordinate: cell no. Growth curves of wild type (O-O, 0-O) and ts2 (O-0, U-m) cells after temperature shift from 39°C (solid symbols) to 33°C (open symbols). The arrows indicate when shift-down was made.
Synchronized cell population was prepared according to the method described by Doida & Okada [IO]. In order to apply their method to our culture conditions, the following modifications were made. Exponentially growing cells were incubated with lO-3 M of thymidine for 8 h at 33°C. Then they were collected by centrifugation and resuspended in the prewarmed fresh medium containing IOe6 M of deoxycytidine. After 5 h, colcemid was added at a final concentration of 0.05 pg/mI and incubated for 5 h. After re-
Fig. I. Abscissa:
were washed, plated in soft agar medium and incubated at 33°C. The developed colonies were tested for growth at 33°C and 39°C. A mutant, ts2, was used for the present study.
2 a 1
0.5 b
Measurement of macromolecular synthesis All the radiochemicals were purchased from Daiichi Pure Chemicals Co., Ltd., Tokyo. Cells were pulselabeled for I h with I &i/ml of [3H]TdR (spec. act. 14.5 Ci/mmole), 1 &i/ml of [3H]uridine (15.0 Ci/m mole), or 0.5 pCi/ml of [Y-Llleucine (204.0 mCi/ mmole). When [3H]uridine was used, unlabeled thymidine and deoxycytidine were added together at IO pg/mI each to reduce the incorporation of uridine into DNA. To measure the incorporation of [Y-L]Ieucine into acid-insoluble fraction, media containing onetenth the normal concentration of leucine were used. Immediately after labeling, the cells were chilled and IOO-fold excess unlabeled precursors were added. They were then applied to the presoaked glass fiber filters (Whatman, type GF/F) and treated successively with phosphate buffered saline (PBS), ice-cold 5% trichloroacetic acid (TCA) and methanol, and the filters were counted as described before [8].
Light microscopic observation and autoradiography At appropriate times, cells were collected and washed twice with PBS. For light microscopic observation, the cells were smeared on slide glasses, dried and Exp Cell Rrs IO0 (1976)
t
0
10
20
30
time (hours); ordinate: rel. radioactivity per cell. Incorooration of (a) 13H1TdR: (b) 13Hluridine; (c) [‘4C-L]I&cine into wild-type (O-0, -0-O) and ts2 (O-0, m-m) cells at 33°C (open symbols) and 39°C (solid symbols). 2x I04~ceIIswere inoculated and cultured for 20 h at 33°C. Then half of cultures were switched to 39°C. After various lengths of time at 33°C and 39”C, radioactive precursors were added for 1 h and radioactivities incorporated into acidinsoluble materials were determined. At the same time cell numbers were counted in a Coulter counter and the incorporation was expressed on a per-cell-basis.
Fig. 2. Abscissa:
A division mutant of L5178Y
A
00'
doubling time of 17-19 h. At 39°C wild type cells grew well with a doubling time of 11 h, whereas ts2 cells ceased to grow after l-2 days and the cell number decreased thereafter. Note that when ts2 cells were cultured at a higher cell density (I x loj cells/ ml) at 39”C, their growth character was identical to the result shown in fig. 1. This property is different from that of the mutant reported by Wittes & Ozer [I 11, which showed density-dependent temperature sensitivity. The plating efficiencies in soft agar at 33°C and 39°C were 102% and 63 % for wild type cells, and 52% and 0.13 % for ts2 cells, respectively.
*
20
299
40
3. Abscissa: time (hours); ordinate: labeling index 6%). Changes in labeling index of wild type (O-O, 0-O) and ts2 (El-O, D-m) cells with time of incubation at 33°C (open symbols) and 39°C (solid symbols). 5x IO4cells were inoculated at time zero and the cultures were incubated at 33°C and 39°C. After various lengths of incubation, I h-pulse labeling of [3H]TdR was performed. The cells which had more than IO silver grains were counted as the cells synthesizing DNA.
Fig.
moving the drug by washing twice with PBS, the cells were used as a synchronized cell population. Under the experimental conditions, cells showed 50-70% synchrony and the duration of Gl, S, G2 and M was 3-4, 9-10, 34 and 0.5-l h, respectively.
RESULTS Cell growth at the permissive and non-permissive temperatures Cell growth of parental strain (wild type) and temperature-sensitive mutant ts2 was measured at the permissive (33°C) and nonpermissive (39°C) temperatures. As shown in tig. 1, at 33°C in liquid medium, wild type cells grew with a doubling time of 16-17 h and ts2 cells grew with a slightly longer
Temperature shift experiment Mutant cells kept at 39°C were shifted down to 33°C at various times and cell growth was measured. As seen in fig. 1, upon shiftdown cells resume growth with a doubling time comparable to that of control after delay of l-3 days, though the recovery of cell growth was retarded as the periods of heat exposure prolonged. This result shows that the mutant cells retain their ability for division even after maintenance at 39°C for as long as 4 days. Macromolecular synthesis at the non-permissive temperature In order to know the effect of temperature on ts2 cells, the synthesis of three types of macromolecules, DNA, RNA and proteins was examined. At various times, wild type and mutant cells (2-3~ lo4 cells/ml) were pulse-labeled with radioactive precursors for 1 h at either 33°C or 39°C. As shown in fig. 2a, at 39°C the incorporation rate of [3H]TdR on a per-cell-basis fell gradually in ts2 cells. However, uptake of [3H]uridine and [‘“Clleucine did not differ between 33°C and 39°C (fig. 2b, c).
300
Shiomi and Sato
Fig. 4. Morphology of ts2 cells cultured at (a) 33°C; (b) 39°C for 20 h; and (c) 40 h. x 1300.
Labeling index The previous experiment (fig. 2~) showed that the incorporation rate of r3H]TdR into acid-insoluble material was affected at 39°C. In order to clarify this phenomenon further, the fraction of cells synthesizing DNA was measured autoradiographically. The appropriate number of cells were incubated at 33°C and half of them were switched to 39°C. At varying times, r3H]TdR was added to the cultures for 1 h and then samples were treated for autoradiography. As shown in fig. 3, the fraction of ts2 cells that were able to synthesize DNA remained constant at 33°C but decreased steadily at 39°C the labeling index of 68% at time zero being reduced to 41% after 36 h. In contrast, no difference was found in the labeling indices in wild type cells even at 36 h between 33°C and 39°C. Thus it is likely that the decrease in thymidine incorporation observed at 39°C in ts2 cells is due to the decreased fraction of cells synthesizing DNA. Morphological changes observed during incubation at 39°C When ts2 cells were grown at 33”C, normal mitotic figures were observed (fig. 4a), but Exp
Cell
Res
IW
(1976)
at 39°C many aberrant mitotic figures and multinucleate cells were found (fig. 4b). In the aberrant mitotic cells, chromosomes appear no longer to be held together by the spindles and are scattered in the cytoplasm. These figures resemble those whose spindle fiber formation is blocked by specific inhibitors such as colchicine. Many multi-
/
A
Fig. 5. Abscissa:
time (hours); ordinate: fraction of cells (%). Changes in mitotic index (O-O, 0-a) and percentage of multinucleate cells (A-A, A-A) in ts2 cells after various periods of incubation at 33°C (open symbols) and 39°C (solid symbols). I x 105cells were inoculated. The arrows indicate when shifts from 39°C to 33°C were made. The mitotic index is expressed as the percentage of cells engaged in mitosis. The cells which had more than one nucleus are scored as multinucleate cells.
A division mutant of L5178Y
301
The number of multinucleate cells increased with time of incubation as well. However, neither mitotic indices nor multinucleate cells changed in ts2 cells incubated at 33°C (fig. 5). When the cells incubated at 39°C were shifted to 33°C at 24 h, the mitotic cell fraction rather increased and the multinucleate cell fraction decreased slowly (fig. 5). Experiments with synchronized cells As can be seen in fig. 5, temperatureinduced mitotic arrest is not apparent until 6 h following shift-up. This result suggests that cells in the late G2 and M phases are not affected by the elevated temperature. Therefore, using the synchronously growing cells we examined which stage in a cell cycle was affected by temperature. Our synchronized population had the
I
I
0
20
I
40
Fig. 6. Abscissa: time (hours); ordinate: fraction of cells (%). (a) Change in mitotic index (O-O, 0-O); (b) percentage of binucleate cells (CL-O, H-W); and
(c) percentage of cells with more than two nuclei (A-A, A-A) in synchronized ts2 cells after various periods of incubation at 33°C (open symbols) and 39°C (solid symbols). After colcemid release cells in the Gl phase were shifted to 39°C (arrows). 0 10
nucleate cells were also found which have two or more normal sized nuclei, or two or more unequally separated lobes of a nucleus (fig. 4~). In order to determine the relation between aberrant mitosis and multinucleation, the number of cells showing these figures was scored at various times of incubation at the non-permissive temperature. As shown in fig. 5, in mutant cells, the mitotic indices began to increase at 6 h after shift to 39”C, reached the maximum of 24% at 20 h and decreased thereafter.
ii-i
0
20
40
time (hours); ordinate: fraction of cells (%). Symbols were the same as in fig. 6. Synchronized cells in the G2 phase were shifted to 39°C (arrows).
Fig. 7. Abscissa:
Exp Cd/Revs 100 (1976)
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mitotic index of 50% at the time of colcemid release. Most of these cells passed through the mitotic phase after 3 h-incubation in the colcemid-free medium, traversed the cell cycle and synchronously entered the second mitotic phase after 14 h (figs 6a, 7a). The cells incubated at 33°C for 3 h and 13 h after release from colcemid block were used as the GI and G2 cell populations, respectively. When cells were shifted up at the Gl phase, the first peak of mitotic indices was much higher than that of the cells cultured at the permissive temperature (fig. 6a). As the mitotic indices decreased, binucleate cells accumulated quickly (fig. 6b) and the increase in the fraction of cells with more than two nuclei followed with some delay (fig. 6~). At 33°C binucleate cells did not accumulate at all. On the other hand, when cells in G2 phase were switched to the non-permissive temperature, the first cell division appeared to occur normally, but the second one was affected, namely, a much higher peak of mitotic indices and the accumulation of binucleate cells (fig. 7a, b). In synchronized wild type cells, no difference in the mitotic indices was observed between 33°C and 39°C. Thus upon shift-up cells in the Gl phase show abnormal mitosis and blocked cytokinesis, while the G2 cells can complete the first, but not the second division. DISCUSSION How do we account for the characteristic morphological changes and growth responses of ts2 cells? Mitotic arrest induced by the restrictive condition appears always to be followed by cytokinetic block in the present mutant. If this is the case, substances affected by temperature would be necessary for both mitosis and cytokinesis. Moreover, experiments with synchronized culture indicate that the temperature-sensiExp CellRcs lOO(l976)
tive stage in ts2 cells is located somewhere from the early Gl through the late G2 phase, though the temperature effect becomes apparent in the M phase. Three temperature-sensitive mutants with a defect in cytokinesis [4,6,7] and one mutant in mitosis [2] have been described. In contrast to these, the present mutant ts2 appears to be defective in both processes of cell division. Drugs such as colchicine and cytochalasin are known to block mitosis and cytokinesis, respectively. But no drugs have so far been reported which inhibit both processes. Considering these findings it is difficult to connect the defective function in ts2 cells with microtubules or microfilaments. Furthermore, the defectiveness is not manifested unless the elevated temperature is given far before the time of its expression. We are currently determining the more precise location of the heat-susceptible stage in the cell cycle in the mutant. The authors thank Drs A. Matsushiro and Y. Nishimune for discussions, Dr K. Mizobuchi for reading the manuscript, and Miss Naoko Hieda for assistance. This work was supported by grants-in-aid for Scientific and Cancer Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Howard, A & Pelt, S R, Heredity 6, suppl. (1953) 261. 2. Wane. R J. Nature 248 (1974) 76. 3. Liskay, R M, J cell physiol 8;1(1974)49. 4. Smith. B J & Wigglesworth, N M, J cell _physiol82 (1973)‘339. -5. Burstin, S J, Meiss, H K & Basilica, C, J cell physiol84 (1974) 397. 6. Thompson, L H & Baker, R M, Methods in cell biology (ed D M Prescott) vol. 6, p. 260. Academic Press, New York (1973). 7. Hatzfeld, J & Buttin, G, Cell 5 (1975) 123. 8. Sato, K & Shiomi, T, Exp cell res 88 (1974) 295. 9. Sato, K, Nature 257 (1975) 813. 10. Doida, Y & Okada, S, Exp cell res 48 (1967) 540. 11. Wittes, R E & Ozer, H L, Exp cell res 80 (1973) 127. Received January 30, 1976 Accepted February 5, 1976