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Mitotic arrest by melatonin
Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research 78 (1973) 314-318
MITOTIC ARREST BY MELATONIN SUMANA
BANERJEE and LYNN MARGULIS
Department of BioIogy, Boston University, Boston, Mass. 02215, USA
SUMMARY We are testing the hypothesis that migration of the newly formed mouth (i.e., oral membranellar band) in stentor is homologous to mitotic chromosomal movement and that both types of movement within single cells depend directly on microtubule elongation. The following compounds synchronously delay the migration of the oral membranellar band as an exponential function of concentration: Colcemid, podophyllotoxin, /I-peltatin and vinblastine. Delay for these compounds can be described by the equation, y = kxn, where y is delay in hours and x is concentration of mitotic spindle inhibitor in moles/l. We discovered that the animal pineal gland hormone, melatonin (5-methoxy n-acetyl tryptamine), also specifically and reproducibly delays oral band regeneration according to an equation of this form. Thus we predicted that melatonin would arrest mitosis. We report here a colchicine-type disruption of the mitotic apparatus in onion root tips by melatonin. Two closely related tryptamine derivatives, n-acetyl serotonin and serotonin were inactive in both the stentor and onion assays: they neither delayed band migration in stentor as an exponential function nor induced mitotic arrest in onion.
Single cells of the heterotrichous ciliate, Stentor coeruleus, can be induced to regenerate their ‘mouths’ in synchrony (that is, their ciliated oral membranellar bands, MB [l-3]. Synchronous regeneration is thus induced in all cells. It involves RNA and protein synthesis, and cilia regeneration to form a new lateral MB (stages O-3 [l-4]) followed by the movement of the newly formed MB to its final position at the anterior end of the cell (stages 4-8 [4]). The MB ‘migrates’ nearly half the length of the cell, approx. 300 pm in some 3-4 h. This slow movement of about 1.5 ,um/min corresponds to the lower end of the range of observed mitotic movement [5]. The MB, which is mature by stage 4 [4], is believed to move by elongation of root fiber microtubules. We postulate that both MB movement and chromosomal movement is directly Exptl Cell Res 78 (1973)
dependent on the elongation of microtubules via the polymerization of tubulin protein into microtubules and have thus tested a range of mitotic spindle inhibitors on band regeneration [6-131. We have found that MB migration is synchronously delayed as an exponential function of concentration of inhibitor for several well-known inhibitors of mitotic spindle function (table 1). These compounds, which have no other deleterious effect on morphogenesis over the physiological range of concentrations, we consider ‘active’ in the stentor assay. As concentrations are increased, corresponding to a maximum for each drug, delay is not increased, rather the cells begin to develop asynchronously, become vacuolated and bleached; at even higher concentrations they die. The sublethal effects are quickly reversible upon removal of the drug. Although
Mitotic arrest by melatonin
Table 1. Delay of oral membranellar mitotic spindle inhibitors
315
in Stentor coeruleus (stage 3) by
band migration
Physiological range ( x 10-OM)
Compound
Reference to activity as mitotic spindle inhibitor
Delay equation
Podophyllotoxina gyze;b . a
WI t171 PI
1.1 x 109 [PI’,’ 6.6 1.9 x 10’2 lox3 [&PI’-8 [Cl””
4 30 0.2
126,271 2.2 x 106 [VI I,0 This paper 5.1 x lOIs [M]3,9
0.8 200
Vinblastineb Melatoni# Griseofulvinb
[18, 261
None
Lowest concentration at which delay can be detected
5
Highest concentration at which delay only is observed
17 957 6: 25
Concentration causing reversible abnormalities
18 100 8 9: 30
Death
21 200 10 20 1000 40
a Computer analysed curves fit by least squares, supercede those fit graphically, see [13]. ’ Computer analysed curves fit by least squares, see [lO-131 for discussion.
delays and abnormalities have been observed for griseofulvin [12], serotonin, n-acetyl serotonin and certain podophyllotoxin derivatives and other compounds [IO, 111, none so far tested but those in table 1 generate a specific exponential delay equation. We confirmed the observation [14] that at certain relative concentrations of Colcemid and melatonin (the animal pineal gland hormone, 5 methoxy n-acetyl tryptamine [15]), the effects of Colcemid were reversed by melatonin, but we found that at higher concentrations the drugs acted synergistically. We then tested melatonin alone on stentor regeneration. To our surprise we found that melatonin also delays MB migration as an exponential function of concentration [ 131. This discovery prompted us to test the effects of melatonin on the mitotic spindle of onion root tips. METHODS Alliurn cepa (obtained at a local grocery) were germinated in the dark in distilled water. When many root tips averaged about 3-5 mm long they were placed in melatonin, serotonin and n-acetyl serotonin (Sigma
Chemical Co., St Louis) and Colcemid (CIBA, Summit, N.J.) solutions, made up in distilled water and enough absolute ethanol, ~0.2 ml, to dissolve. The roots were incubated and sample preparations made at 3 h, 24 h and 8 days when the experiment was terminated. About 3 mm of tip was severed with a razor blade at about 1 p.m., known to be a time of high mitotic activity. The tips were fixed in 3 : 1 ethanol/acetic acid for at least 1 h. They were removed to 45 % acetic acid for 5 min and stained in 1% lactic aceto-orcein (9 parts) to 1 part concentrated HCI (9 : 1, see [16]) after they were heated in the stain solution and gently shaken to soften the tissue. The best preparations were made from roots stained 24 h in 9 : 1 and squashed on slides in 1 drop 1 % lactic acetoorcein. The preparations were made semi-permanent by sealing the coverslips with paraffin. The entire melatonin experiment was repeated with a second set of 20-30 root tips from onions obtained from a different source; the results were the same.
RESULTS We found root tips grown in melatonin had the typical stubby appearance of those grown in Colcemid (fig. 1); after 3 h in 8 x lo-” M no normal metaphases or anaphases were seen(fig. 2). In 100% of the preparations the mitotic spindle was disrupted; typical ‘colchitine-mitosis’ metaphases with the chromosomesin ‘ski configurations’ (attached at the centromere, see [17]) were the only abnormalExptl Cell Res 78 (1973)
3 I6
Sumana Banerjee and Lynn Margulis Table 2. Effect of tryptamines on the mitotic index in Allium at 24 h Treatment
Concentration (moles/l)
% cells in mitosisa
Control n-Acetyl serotonin Serotonin Melatonin*
Distilled water 6.4 x 1O-3 9.0 x IO-4 8.0 x lO-a
9.98 kO.51 3.12kO.24 3.52kO.31 8.40&0.12
Fig. I. Onion roots grown for 8 days in melatonin (8 x 1O-4 M) (right) control.
a Mitoses in five fields were counted and averaged; phase contrast microscopy x 1 000. Each entry represents the average of these from five different root tips. The number of prophase > metaphases > telophases > anaphases and all stages were normal except in melatonin. b No anaphases or telophases observed; all cells in mitosis were arrested in ‘metaphase’.
ities observed. After 24 h, polyploid nuclei progressively became more abundant; after 8 days the tissue damage precluded measurements of mitotic indices. Although the mitotic index of cells grown in melatonin was comparable to that of the control, mitotic figures of cells grown in melatonin were indistinguishable from those grown in 2 x 1O-4 M Colcemid. For some unknown reason the epidermal layer of cells became progressively browner in the solutions of all 3 tryptamine derivatives (fig. l), but in other respects the results of treatment with serotonin and IZacetyl serotonin were entirely different. No spindle disruption was observed and the
relative proportions of mitotic stages were unaltered. At low concentrations these tryptamine derivatives had no effect on the growing root tips; at higher concentrations they lowered the mitotic index (table 2). Neither metaphase arrest nor polyploidy was observed in any root treated with these compounds. The length and the number of root tips germinated were unaltered in serotonin and nacetyl serotonin. This is in contrast to all roots growing in melatonin which were significantly shorter and stubbier than controls and had fewer lateral hairs. Our inability to quantify the delay induced by griseofulvin in stentor [lo, 1l] led us to
Fig. 2. Melatonin induced mitotic arrest; squashes from cells 3 h in 8 x 1O-4 M (left) control in anaphase ( x 1 000). Exptl Cell Res 78 (1973)
Mitotic arrest by melatonin 317 retest the effect of this compound on Allium root tips. After 3 and 24 h in 1 X 10e5 M griseofulvin we observed nonspecific spindle disruption. Chromosome damage was seen and most cells were blocked in an abnormal looking prophase. The precise colchicine-like mitotic arrest was not observed; our observations were more like those seen in Aspergillus nidulans [25]. Because of the inability to clearly distinguish mitotic stages we felt that measurements of the mitotic index of griseofulvin-treated cells were meaningless. Deysson [I 81 simply counted these abnormal cells as prophases. Thus our results were quite comparable to those of Deysson although our conclusion that griseofulvin differs somewhat from colchicine in its mechanism of action contradicts his interpretation. This discovery of mitotic arrest induced by melatonin is consistent with our hypothesis that stentor MB migration and chromosome movement are homologous processes. Most investigators have used these drugs in vivo at a concentration sufficient to induce mitotic arrest. We suggest that reversible delay of chromosome movement as a function of concentration be measured at a series of low concentrations of these inhibitors in live cells. We predict such observations will generate delay curves with the same exponents and constants that we have measured (table 1). The concept [19] that all of the inhibitors in the table act by binding to tubulin and preventing its assembly into microtubules is consistent with recent biochemical results [20, 211 as well as the inactivity reported for griseofulvin [20, 221. It strengthens one classical concept of force generation during mitosis [23]: chromosomes move because they are attached to the spindle (i.e., polymerizing microtubule protein) that grows inbetween [24]. We are testing a current hypothesis that activity (e.g., metaphase arrest and delay of band migration as an exponential
function of concentration) requires a methoxy group on an aromatic ring approximately 7 A away from an electronegative atom that can hydrogen bond to the receptor protein (T. N. Margulis. Pers. comm., see [lo, 111for discussion). Our results therefore suggest a testable hypothesis: that the mechanism of action of melatonin in higher animals involves hydrogen bonding to receptor tubulin proteins. We acknowledge the aid of T. N. Margulis, J. K. Kelleher. S. Proost. M. Winston. and C. LaRueHonig in collecting ‘and analysing’data presented in Table 1. We thank NASA NGR-004-025 for research support.
REFERENCES 1. Tatar, V, The biology of Stentor. Pergamon Press, New York (1961). 2. - Trans Am microsc sot 87 (1968) 297 3. Burchill, B R, J exptl zoo1 167 (1968) 427. 4. Paulin, J J & Bussey, J, J protozool 18 (1971) 201. 5. Nicklas, R B, Mitosis in advances in cell biology (ed D M Prescott, L Goldstein & E H McConkey) vol. 2, p. 1. Appleton-Century-Crofts, New York (1971). 6. Neviackas, J & Margulis, L, J protozool 16 (1969) 165. I. Banerjee, S & Margulis, L, Nature 224 (1969) 180. 8. Makrides, E, Banerjee, S, Handler, L & Margulis,
L, J protozool17 (1970) 548. 9. Banerjee, S & Margulis, L, Cancer chemother
rep 55 (1971) 531. 10. Margulis, L, Ann rev cytol 34 (1972) 333.
11. Propst, S, Banerjee, S, Kelleher, J K & Margulis, L, Cancer chemother rep 56 (1972). In press. 12. Margulis, L, Neviackas, J A & Banerjee, S, J protozool 16 (1969) 660. 13. Banerjee, S, Kerr, V, Winston, M, Kelleher, J K & Margulis, L, 3 protozool 19 (1972) 108. 14. Malawista, S E, Sato, H & Inoue, S, Biol bull 129 (1965) 414. 15. Wurtman, R J, Axelrod, J & Kelly, D E, The pineal. Academic Press, New York (1968). 16. Sharma, A & Sharma, A, Chromosome techniques, theory and practice. Butterworths, Washington D.C. (1965). 17. Eigsti, H & Dustin, P, Colchicine in chemistry, medicine, and agriculture. University of Iowa Press, Ames, Iowa (1955). 18. Deysson, G, Ann pharmaceut franc 22 (1964) 2. Borisy, G G &Taylor, E, 3 cell biol 35 (1967) 535. :;: Wilson, L, Biochemistry 9 (1971) 4999. 21. Stephens, R E, Microtubules in biological macromolecules (ed S N Timasheff & C D Fasman). Academic Press, New York (1971). Exptl Cell Res 78 (1973)
3 18 Sumana Banerjee and Lynn Margulis 22. Williams, J A & Wolff, J, Proc natl acad sci US 67 (1970) 1901. 23. Cleveland, L R, Mem Am acad arts sci 17 (1934) 185. 24. Margulis, L. In preparation (1973). 25. Crackower, S H B, Canad j microbial 18 (1972) 683.
Exptl Cell Res 78 (1973)
26. Malawista, S E, Sato, H & Bensch, K G, Science 160 (1968) 770. 27. Johnson, I S, Exptl cell res 20 (1960) 198. 28. Kelly, M G & Hartwell, J L, J natl cancer inst 14 (1954) 967. Received October 31, 1972.
Experimental Cell Research 78 (1973) 314-318
MITOTIC ARREST BY MELATONIN SUMANA
BANERJEE and LYNN MARGULIS
Department of BioIogy, Boston University, Boston, Mass. 02215, USA
SUMMARY We are testing the hypothesis that migration of the newly formed mouth (i.e., oral membranellar band) in stentor is homologous to mitotic chromosomal movement and that both types of movement within single cells depend directly on microtubule elongation. The following compounds synchronously delay the migration of the oral membranellar band as an exponential function of concentration: Colcemid, podophyllotoxin, /I-peltatin and vinblastine. Delay for these compounds can be described by the equation, y = kxn, where y is delay in hours and x is concentration of mitotic spindle inhibitor in moles/l. We discovered that the animal pineal gland hormone, melatonin (5-methoxy n-acetyl tryptamine), also specifically and reproducibly delays oral band regeneration according to an equation of this form. Thus we predicted that melatonin would arrest mitosis. We report here a colchicine-type disruption of the mitotic apparatus in onion root tips by melatonin. Two closely related tryptamine derivatives, n-acetyl serotonin and serotonin were inactive in both the stentor and onion assays: they neither delayed band migration in stentor as an exponential function nor induced mitotic arrest in onion.
Single cells of the heterotrichous ciliate, Stentor coeruleus, can be induced to regenerate their ‘mouths’ in synchrony (that is, their ciliated oral membranellar bands, MB [l-3]. Synchronous regeneration is thus induced in all cells. It involves RNA and protein synthesis, and cilia regeneration to form a new lateral MB (stages O-3 [l-4]) followed by the movement of the newly formed MB to its final position at the anterior end of the cell (stages 4-8 [4]). The MB ‘migrates’ nearly half the length of the cell, approx. 300 pm in some 3-4 h. This slow movement of about 1.5 ,um/min corresponds to the lower end of the range of observed mitotic movement [5]. The MB, which is mature by stage 4 [4], is believed to move by elongation of root fiber microtubules. We postulate that both MB movement and chromosomal movement is directly Exptl Cell Res 78 (1973)
dependent on the elongation of microtubules via the polymerization of tubulin protein into microtubules and have thus tested a range of mitotic spindle inhibitors on band regeneration [6-131. We have found that MB migration is synchronously delayed as an exponential function of concentration of inhibitor for several well-known inhibitors of mitotic spindle function (table 1). These compounds, which have no other deleterious effect on morphogenesis over the physiological range of concentrations, we consider ‘active’ in the stentor assay. As concentrations are increased, corresponding to a maximum for each drug, delay is not increased, rather the cells begin to develop asynchronously, become vacuolated and bleached; at even higher concentrations they die. The sublethal effects are quickly reversible upon removal of the drug. Although
Mitotic arrest by melatonin
Table 1. Delay of oral membranellar mitotic spindle inhibitors
315
in Stentor coeruleus (stage 3) by
band migration
Physiological range ( x 10-OM)
Compound
Reference to activity as mitotic spindle inhibitor
Delay equation
Podophyllotoxina gyze;b . a
WI t171 PI
1.1 x 109 [PI’,’ 6.6 1.9 x 10’2 lox3 [&PI’-8 [Cl””
4 30 0.2
126,271 2.2 x 106 [VI I,0 This paper 5.1 x lOIs [M]3,9
0.8 200
Vinblastineb Melatoni# Griseofulvinb
[18, 261
None
Lowest concentration at which delay can be detected
5
Highest concentration at which delay only is observed
17 957 6: 25
Concentration causing reversible abnormalities
18 100 8 9: 30
Death
21 200 10 20 1000 40
a Computer analysed curves fit by least squares, supercede those fit graphically, see [13]. ’ Computer analysed curves fit by least squares, see [lO-131 for discussion.
delays and abnormalities have been observed for griseofulvin [12], serotonin, n-acetyl serotonin and certain podophyllotoxin derivatives and other compounds [IO, 111, none so far tested but those in table 1 generate a specific exponential delay equation. We confirmed the observation [14] that at certain relative concentrations of Colcemid and melatonin (the animal pineal gland hormone, 5 methoxy n-acetyl tryptamine [15]), the effects of Colcemid were reversed by melatonin, but we found that at higher concentrations the drugs acted synergistically. We then tested melatonin alone on stentor regeneration. To our surprise we found that melatonin also delays MB migration as an exponential function of concentration [ 131. This discovery prompted us to test the effects of melatonin on the mitotic spindle of onion root tips. METHODS Alliurn cepa (obtained at a local grocery) were germinated in the dark in distilled water. When many root tips averaged about 3-5 mm long they were placed in melatonin, serotonin and n-acetyl serotonin (Sigma
Chemical Co., St Louis) and Colcemid (CIBA, Summit, N.J.) solutions, made up in distilled water and enough absolute ethanol, ~0.2 ml, to dissolve. The roots were incubated and sample preparations made at 3 h, 24 h and 8 days when the experiment was terminated. About 3 mm of tip was severed with a razor blade at about 1 p.m., known to be a time of high mitotic activity. The tips were fixed in 3 : 1 ethanol/acetic acid for at least 1 h. They were removed to 45 % acetic acid for 5 min and stained in 1% lactic aceto-orcein (9 parts) to 1 part concentrated HCI (9 : 1, see [16]) after they were heated in the stain solution and gently shaken to soften the tissue. The best preparations were made from roots stained 24 h in 9 : 1 and squashed on slides in 1 drop 1 % lactic acetoorcein. The preparations were made semi-permanent by sealing the coverslips with paraffin. The entire melatonin experiment was repeated with a second set of 20-30 root tips from onions obtained from a different source; the results were the same.
RESULTS We found root tips grown in melatonin had the typical stubby appearance of those grown in Colcemid (fig. 1); after 3 h in 8 x lo-” M no normal metaphases or anaphases were seen(fig. 2). In 100% of the preparations the mitotic spindle was disrupted; typical ‘colchitine-mitosis’ metaphases with the chromosomesin ‘ski configurations’ (attached at the centromere, see [17]) were the only abnormalExptl Cell Res 78 (1973)
3 I6
Sumana Banerjee and Lynn Margulis Table 2. Effect of tryptamines on the mitotic index in Allium at 24 h Treatment
Concentration (moles/l)
% cells in mitosisa
Control n-Acetyl serotonin Serotonin Melatonin*
Distilled water 6.4 x 1O-3 9.0 x IO-4 8.0 x lO-a
9.98 kO.51 3.12kO.24 3.52kO.31 8.40&0.12
Fig. I. Onion roots grown for 8 days in melatonin (8 x 1O-4 M) (right) control.
a Mitoses in five fields were counted and averaged; phase contrast microscopy x 1 000. Each entry represents the average of these from five different root tips. The number of prophase > metaphases > telophases > anaphases and all stages were normal except in melatonin. b No anaphases or telophases observed; all cells in mitosis were arrested in ‘metaphase’.
ities observed. After 24 h, polyploid nuclei progressively became more abundant; after 8 days the tissue damage precluded measurements of mitotic indices. Although the mitotic index of cells grown in melatonin was comparable to that of the control, mitotic figures of cells grown in melatonin were indistinguishable from those grown in 2 x 1O-4 M Colcemid. For some unknown reason the epidermal layer of cells became progressively browner in the solutions of all 3 tryptamine derivatives (fig. l), but in other respects the results of treatment with serotonin and IZacetyl serotonin were entirely different. No spindle disruption was observed and the
relative proportions of mitotic stages were unaltered. At low concentrations these tryptamine derivatives had no effect on the growing root tips; at higher concentrations they lowered the mitotic index (table 2). Neither metaphase arrest nor polyploidy was observed in any root treated with these compounds. The length and the number of root tips germinated were unaltered in serotonin and nacetyl serotonin. This is in contrast to all roots growing in melatonin which were significantly shorter and stubbier than controls and had fewer lateral hairs. Our inability to quantify the delay induced by griseofulvin in stentor [lo, 1l] led us to
Fig. 2. Melatonin induced mitotic arrest; squashes from cells 3 h in 8 x 1O-4 M (left) control in anaphase ( x 1 000). Exptl Cell Res 78 (1973)
Mitotic arrest by melatonin 317 retest the effect of this compound on Allium root tips. After 3 and 24 h in 1 X 10e5 M griseofulvin we observed nonspecific spindle disruption. Chromosome damage was seen and most cells were blocked in an abnormal looking prophase. The precise colchicine-like mitotic arrest was not observed; our observations were more like those seen in Aspergillus nidulans [25]. Because of the inability to clearly distinguish mitotic stages we felt that measurements of the mitotic index of griseofulvin-treated cells were meaningless. Deysson [I 81 simply counted these abnormal cells as prophases. Thus our results were quite comparable to those of Deysson although our conclusion that griseofulvin differs somewhat from colchicine in its mechanism of action contradicts his interpretation. This discovery of mitotic arrest induced by melatonin is consistent with our hypothesis that stentor MB migration and chromosome movement are homologous processes. Most investigators have used these drugs in vivo at a concentration sufficient to induce mitotic arrest. We suggest that reversible delay of chromosome movement as a function of concentration be measured at a series of low concentrations of these inhibitors in live cells. We predict such observations will generate delay curves with the same exponents and constants that we have measured (table 1). The concept [19] that all of the inhibitors in the table act by binding to tubulin and preventing its assembly into microtubules is consistent with recent biochemical results [20, 211 as well as the inactivity reported for griseofulvin [20, 221. It strengthens one classical concept of force generation during mitosis [23]: chromosomes move because they are attached to the spindle (i.e., polymerizing microtubule protein) that grows inbetween [24]. We are testing a current hypothesis that activity (e.g., metaphase arrest and delay of band migration as an exponential
function of concentration) requires a methoxy group on an aromatic ring approximately 7 A away from an electronegative atom that can hydrogen bond to the receptor protein (T. N. Margulis. Pers. comm., see [lo, 111for discussion). Our results therefore suggest a testable hypothesis: that the mechanism of action of melatonin in higher animals involves hydrogen bonding to receptor tubulin proteins. We acknowledge the aid of T. N. Margulis, J. K. Kelleher. S. Proost. M. Winston. and C. LaRueHonig in collecting ‘and analysing’data presented in Table 1. We thank NASA NGR-004-025 for research support.
REFERENCES 1. Tatar, V, The biology of Stentor. Pergamon Press, New York (1961). 2. - Trans Am microsc sot 87 (1968) 297 3. Burchill, B R, J exptl zoo1 167 (1968) 427. 4. Paulin, J J & Bussey, J, J protozool 18 (1971) 201. 5. Nicklas, R B, Mitosis in advances in cell biology (ed D M Prescott, L Goldstein & E H McConkey) vol. 2, p. 1. Appleton-Century-Crofts, New York (1971). 6. Neviackas, J & Margulis, L, J protozool 16 (1969) 165. I. Banerjee, S & Margulis, L, Nature 224 (1969) 180. 8. Makrides, E, Banerjee, S, Handler, L & Margulis,
L, J protozool17 (1970) 548. 9. Banerjee, S & Margulis, L, Cancer chemother
rep 55 (1971) 531. 10. Margulis, L, Ann rev cytol 34 (1972) 333.
11. Propst, S, Banerjee, S, Kelleher, J K & Margulis, L, Cancer chemother rep 56 (1972). In press. 12. Margulis, L, Neviackas, J A & Banerjee, S, J protozool 16 (1969) 660. 13. Banerjee, S, Kerr, V, Winston, M, Kelleher, J K & Margulis, L, 3 protozool 19 (1972) 108. 14. Malawista, S E, Sato, H & Inoue, S, Biol bull 129 (1965) 414. 15. Wurtman, R J, Axelrod, J & Kelly, D E, The pineal. Academic Press, New York (1968). 16. Sharma, A & Sharma, A, Chromosome techniques, theory and practice. Butterworths, Washington D.C. (1965). 17. Eigsti, H & Dustin, P, Colchicine in chemistry, medicine, and agriculture. University of Iowa Press, Ames, Iowa (1955). 18. Deysson, G, Ann pharmaceut franc 22 (1964) 2. Borisy, G G &Taylor, E, 3 cell biol 35 (1967) 535. :;: Wilson, L, Biochemistry 9 (1971) 4999. 21. Stephens, R E, Microtubules in biological macromolecules (ed S N Timasheff & C D Fasman). Academic Press, New York (1971). Exptl Cell Res 78 (1973)
3 18 Sumana Banerjee and Lynn Margulis 22. Williams, J A & Wolff, J, Proc natl acad sci US 67 (1970) 1901. 23. Cleveland, L R, Mem Am acad arts sci 17 (1934) 185. 24. Margulis, L. In preparation (1973). 25. Crackower, S H B, Canad j microbial 18 (1972) 683.
Exptl Cell Res 78 (1973)
26. Malawista, S E, Sato, H & Bensch, K G, Science 160 (1968) 770. 27. Johnson, I S, Exptl cell res 20 (1960) 198. 28. Kelly, M G & Hartwell, J L, J natl cancer inst 14 (1954) 967. Received October 31, 1972.