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Action of nocodazole on the mechanisms of segmentation mitosis
Cell Biology International
Reports, Vol. 1, No. 6, 1977
503
IXZLTON OF NOCODAZCGE ON THE ME(33ANBG OF SEGWNIATION'MITCSIS. Paul SHNTEIN (Laboratoire d'Histologie et d%&yologie FACULTEDEI%!DECINS- 3406OMXFP~CEDEX (France) Segmentatianmitosesin~~ianeggscanbetreatedbya spindle inhibitor atanyphase of the cell cycle. Wehave conside redtheappearancleofthef~st~pi~~p~~ast~t ; 45 to 55 minutes later themtaphase of the secondmitosisocc&s and,atabout9Ominutes, telcphase. Cm the other hand the.picentriolar material, Wch constitutes the large centrcspher% (up to 20 pin diameter atprcsr&aphase),mdergoes characteristic changes during the cell cycle @TFSand SENI'EIN, 1977). -lHbenzimidaNoccdazok, methyl I 5 -(2 thienykarbony~) zole - 2Y - l]carb~te,isanantimitaticfllbstance~chmakes microtubules disappear asdoescolchicine (DE-and toll., 1975-1977). Its effects m the achrcmatic apparatus of the early s~tatianmitoses~estudiedinc~dertodetermineifthey arethesmeasthoseofcolchicine. Cleaving eggs of Tritums he1vet.icusPaz.w~~ incubated in water soluticm of nocodazole 0,75 ~10'~ M (previously dissolved in dimethylsulfoxide) during 45 or 9Ominutes (about1/2 and1 cell cycle respectively) at 19' C. The eggs wfxe fixed, serially sectimedandstainedby Curtis's technique (SENTEIN, 1976). Final ccmentraticm of L%GO(l/1332) is without any effect. Noccdazole was kindly supplied by Dr. DE HRXWDER (labmatory of Oncology, JAFSSENPharmaceutics Research Laboratories, D 2430 BEERSEHelgimtj F?ESWS to lo/ Effects, after 45 minutes, of a tieatmnt Ixz@minq& Themetaphaseoccumdataboutthesmetixeasinthe mtreatedeggs. Aswith the other spindle inhibitorsactingin segmntaticm mitoses, the furrow was absent, reduced or irregular Inthe mtreatedeggs atmataphase the @ndlefi.bresme inaregulararr~andtbeasters,whi~didnat~andintothe vitellinecytoplasm,~e~o~~bya~iastralne~rk (fig. 1 arrmheads). Fig. 1 - untreated metaphase. Fig. 2, 4, 5 - Nxodazole 45 min. Treated
mataphase.
.
504
Cell Biology
lnterna tional Reports,
Vol. I, No. 6, 19 77
Cell Biology International
In the treated
Reports, Vol. 1, No. 6, 1977
eggs (fig.
505
2, 4 ard 5) me&phases were alnor-
ma1 : A) For 11 metaphases, 10 of the times, free of astral fibres (canpletely blocked), surrdd notbyaperiastral fibrillar material less strong lne??&d~ Y s than the centrospheres themselves (fig. 2,
the centrospheres were they were generally !%eab%!%i%o&phtol 4, catpared to fig. 1).
B) Theywere separated franthewt&i by a space without fibres, of variable width 16 p in fig. 4 ard 5 r in fig. 5).
ends of the spirdle (10 /9" in fig. 2,
C) Their structure~sdifferentframthatof a) granular, without peripheral con&nsation granular, with peripheral cor-densation (fig. thick granules with lacunes in between (fig.
thenocmalones: (fig. 2), b) finely 4), or c) formed of 5).
~ehavenotobserved inthis e.xperimentspMical,dense, strongly stained, gllDoth surfaced (cunpletely "blocked") centrosphereS. D) Thespindle7n~sreduced inlength,butnot inwidth,ard only formed by theeguatorialpartof its fibres. Thesefibres were sinuous axxl in irregular array. The conservation of the quatarialpartof the spirdle,whileitspolar parts aredestroyed, remirds us of the separation frcan the spindle of the regressive asters after action of a derivative of colchicine (SIXCEIN, 1965), of chlaralh@rate (SENI!EINar-d AT%, 1974) or of other inhibitors, whenthetimeoftrealnl&zwasshort. E) Thechranosanes,moreorless normal in structure,were notdiqersed inthecytoplasmaswithcolchicine,butstabiliz~ in the equatcrial region ard slightly oblique with respect to the equatorial plane. Theseeffectsof nccodazolea~ earlier than tlmse of colchicine in the same cells. F) We have studied 17 ~ophases in so-treated eggs, in 15, centrosphereswere r-&visiblewitha pMxm.icroscopeard 2 did shw small blocked centrospheres. 2°/Effectsof a 45minutetrea~tbeginnirq 45mjnutesafterto (suppossa eixneof'~~se), Lln~ofdeL?to Stlid~ the effects oFmcodazole at'tekghase (rmmally about 9Ominutes). Thecentrosphereswerea littledenser ardmeheavily stained thannormally. Sane,astral fibreswarepresent, butfwer ard tshrter than rormally (fig. 3, 6, 7, 8). Then these centrospheseswerenotcanpletelyblocked. There canbe eit&r a single
506
Cell Biology
International
Reports,
Vol. 1, No. 6, 19 77
transversally lenghtened centrosphere (fig. 3, 6) or TV centrospheres (fig. 7, 8) more or less separated one from the other, according to thetimewhentheblcckingcccurred.. Whenthe treated eggs containedmetaphases or anaphases, the centrosphereswereblocked (9mitoses) or absent (2mitoses).When they contained telophase, 4 mitoses were blocked and 23 only partially. 3’/
Effects of a 45 minute treatment beginning at to with ensuing recoveryduring 9ominutes.
A) The nuclei, free of centrospheres and fibres (apolar nuclei), were not in the central region of the blastaneres, but nearer tothecortexard theanimalpole, always embedded inanon vitelline cytoplasn inthe formof a cupola, as if they had carried with then the surrourding hyaloplasm. The process of depolarization continusd innormalmedium. B) The structure of nuclei was always telophasic (with karyomeres) or telo-pophasic. There were usually tx~ nuclei for an hyaloplasmic zone, in scme cases they had fused, C) In the central part of sane nuclei chratrxanes were not ccqletely transformed into chrwsanal vesicles, while this transformation was made at their periphary. Itisnotewxthythat,atthecorrespoxxUngtime,untreated eggswereatthemiddleof thefourthcycle (4metaphases) at the end of the third (2 telophases). 4”/
Effectsof atreatmentduring toardduring 9ominutes.
ard not
the secordmitisisbeginniqat
Thecentrosphereswereccmpletelyblocl&,nrxe~eneXs after 45 minutes, but their structure rsfnaird more or less granular. Scxne"rosette" nuclei appeared (fig. 9). than
5'/ Action of the same concentration stage of segmentation.
during 90 minutes at any
In sane morulas, centered nuclei ("rosettes") could be observed (fig. 9). The centrospheres were ccfrpletely blocked at any phase of mitosis : for instance at mataphase (fig. lo), telophase or telo-wophase (fig. 11) ; they were dense, strongly staindard freeof f&es, scxnetimeswithaless stained center ("enpty centrospheres) (fig. 12). The closer the cycle came to its erd, the smaller the centrospheres.
Cell Biology
International
Reports,
Vol. 1, No. 6, 19 7 7
508
Cell Biology International
Repotts, Vol. 1, No. 6, 1977
lo/ Atmetaphase of the secord mitisis, the sianiltanexs disap pearanceof theastral fibresard of the polar parts of the spindle fibres,whiletheir equatorialpartsare~e~~,mustbe~~inedbyadifference insensitivity of thesets+otypesof fibres, which are of different origin (respectively as-led by the pericentriolar ardbythekinetocboreorganizingcenters). Irde& the former arer&rmallyassembled beforethelatter. Theastralfibres ~thepolarpartsafthespirdlef~esbehaveinthesame~y. Intheabaeof the form&r, theequatiialpartsof the spirdle fibres aaulii mtbeorientior stretched. 2”/ Atmetaphasetherecognisable centrosphereswit.l-outastral fibres, blocked after a 45 minute trealment, always appearmore orlessgrarxUr.This typeof centrospherewasobs~ed at the beginning of the action of saturated qutiline (see SDJI!EIN 1970 fig. 13 a 17, 27 and 46). EUt after a 90 minute treatment, dense, lxmngeneous, srooth-surfacedcentrosphereswerefaund. The former type seems lmbe thefirststepof the latter.
3'/ The fact that the astral fibres do r& canpletely disappear at the end of telophase after 45 minutes, suggests that rxxodazole destsaysthanlessquickly~theastersareexpansivethan whentheybeccme smaller. 4’/ Thecentrospheresarerarelyvisible atwophaseafter action of rrxcdazole. In similar cases, when the cycle may be considered as relatively slowed down, this effect could have begun at the Theabsenzeofpericentriolar preceding anaphaseortelophase. materialcculdbedueeither toaninhibitionof themicrotubule assembly at anaphase or to an enharrzenent of their disassgnbly attelophase.
5'/ The centrospheres are n-ore qletely blocked after.a longer treatment (9Ominutes), lxtcanpletelydisappear, as well as the fibres, 9Ominutesafter the erd of a 45minutetreatment. This ~ndoesnot~thatnxodazolehas inanywayanirre versible action,butprobablyreflectsthe solidbindingof the drug tomicrotubule subunits. 6'/ All the observations made on segmentation mitoses lead to the conclusion that noccdazole has not the same effects as colchicine : it does not make centrospheres disappear, they are "blocked" as with quinoline. The question still staMs : is the difference a qualitative one or is it a cons-e of the rapid action of noccdazoleascanpared toother spindle inhibitors? Fig. 3, 6, 7, 8 -Same treatmtm&telophase. Fig. 9 to 12 - Noccdazole 1 H 30 min.
Ceil Biology International
509
Reports, Vol. 1, NO. 6, 1977
ATES, Y. et SENlXN, P. (1977) Centrospheres ad cellular in the cleaving eggs of Pleur&eles waltlii Michah. Biologiecellulaire, sous Fesse.
cycle
DE BRABANDER, M., VAN DE VElRE, R., AEliTS, P., GZJENSG., axd DE CREE, J. (1975) Orkxdazole EDRZRS, M., DESPm,L. (R. 17934) : anewanticarxzerdrug interferingwithmicrotubules. Effect on neoplastic cells cultured in vitro and 2 vivo.Microtubulesardmicrotubule inhibitors, eds.M. BXGEBS et. DE Bl?AmNDER,509-521. DE BRAIBNDEEt,M., DEMEY,J., SONIAU, M. and GWENS, G. (1977) Ultrastructural ~enical distribution of tubulin incultured cells treatedwithmicrotubule inhibitors.Cell Biology Intern. Reports, L, 177-183. SENTETN,P. (1965) Ananalies des tiles obmes par l'action du sel&ium sur les mitoses de segmentation chez des es&&es r&sistantes. Canparaison avec l'effet de l'&hylamino%sac&ylcolchicide. C. R. Acad. Sci. Paris, 260, 5904-5906. Sm, P. (1970) Action de la quinoline sur les mitoses de segmentation des oeufs d'Urod~les : le blocage de la centrosphere. Chrmsc~~ (B-l.), 32, 97-134. S-IN, P. (1976) Methods of fixing, sectioning ard staining Amphibianeggs for cytological study.MicroscopicaActa,7& 427-438.
SENPEIN, P. et ATES, Y. (1974) Action de l'hydrate de chloral sur les mitoses de segmentation de l'oeuf de Pleurcdele. Etude cytologique et ultrastructurale. Chratosana (Berl.), 45, 215-244.
Reports, Vol. 1, No. 6, 1977
503
IXZLTON OF NOCODAZCGE ON THE ME(33ANBG OF SEGWNIATION'MITCSIS. Paul SHNTEIN (Laboratoire d'Histologie et d%&yologie FACULTEDEI%!DECINS- 3406OMXFP~CEDEX (France) Segmentatianmitosesin~~ianeggscanbetreatedbya spindle inhibitor atanyphase of the cell cycle. Wehave conside redtheappearancleofthef~st~pi~~p~~ast~t ; 45 to 55 minutes later themtaphase of the secondmitosisocc&s and,atabout9Ominutes, telcphase. Cm the other hand the.picentriolar material, Wch constitutes the large centrcspher% (up to 20 pin diameter atprcsr&aphase),mdergoes characteristic changes during the cell cycle @TFSand SENI'EIN, 1977). -lHbenzimidaNoccdazok, methyl I 5 -(2 thienykarbony~) zole - 2Y - l]carb~te,isanantimitaticfllbstance~chmakes microtubules disappear asdoescolchicine (DE-and toll., 1975-1977). Its effects m the achrcmatic apparatus of the early s~tatianmitoses~estudiedinc~dertodetermineifthey arethesmeasthoseofcolchicine. Cleaving eggs of Tritums he1vet.icusPaz.w~~ incubated in water soluticm of nocodazole 0,75 ~10'~ M (previously dissolved in dimethylsulfoxide) during 45 or 9Ominutes (about1/2 and1 cell cycle respectively) at 19' C. The eggs wfxe fixed, serially sectimedandstainedby Curtis's technique (SENTEIN, 1976). Final ccmentraticm of L%GO(l/1332) is without any effect. Noccdazole was kindly supplied by Dr. DE HRXWDER (labmatory of Oncology, JAFSSENPharmaceutics Research Laboratories, D 2430 BEERSEHelgimtj F?ESWS to lo/ Effects, after 45 minutes, of a tieatmnt Ixz@minq& Themetaphaseoccumdataboutthesmetixeasinthe mtreatedeggs. Aswith the other spindle inhibitorsactingin segmntaticm mitoses, the furrow was absent, reduced or irregular Inthe mtreatedeggs atmataphase the @ndlefi.bresme inaregulararr~andtbeasters,whi~didnat~andintothe vitellinecytoplasm,~e~o~~bya~iastralne~rk (fig. 1 arrmheads). Fig. 1 - untreated metaphase. Fig. 2, 4, 5 - Nxodazole 45 min. Treated
mataphase.
.
504
Cell Biology
lnterna tional Reports,
Vol. I, No. 6, 19 77
Cell Biology International
In the treated
Reports, Vol. 1, No. 6, 1977
eggs (fig.
505
2, 4 ard 5) me&phases were alnor-
ma1 : A) For 11 metaphases, 10 of the times, free of astral fibres (canpletely blocked), surrdd notbyaperiastral fibrillar material less strong lne??&d~ Y s than the centrospheres themselves (fig. 2,
the centrospheres were they were generally !%eab%!%i%o&phtol 4, catpared to fig. 1).
B) Theywere separated franthewt&i by a space without fibres, of variable width 16 p in fig. 4 ard 5 r in fig. 5).
ends of the spirdle (10 /9" in fig. 2,
C) Their structure~sdifferentframthatof a) granular, without peripheral con&nsation granular, with peripheral cor-densation (fig. thick granules with lacunes in between (fig.
thenocmalones: (fig. 2), b) finely 4), or c) formed of 5).
~ehavenotobserved inthis e.xperimentspMical,dense, strongly stained, gllDoth surfaced (cunpletely "blocked") centrosphereS. D) Thespindle7n~sreduced inlength,butnot inwidth,ard only formed by theeguatorialpartof its fibres. Thesefibres were sinuous axxl in irregular array. The conservation of the quatarialpartof the spirdle,whileitspolar parts aredestroyed, remirds us of the separation frcan the spindle of the regressive asters after action of a derivative of colchicine (SIXCEIN, 1965), of chlaralh@rate (SENI!EINar-d AT%, 1974) or of other inhibitors, whenthetimeoftrealnl&zwasshort. E) Thechranosanes,moreorless normal in structure,were notdiqersed inthecytoplasmaswithcolchicine,butstabiliz~ in the equatcrial region ard slightly oblique with respect to the equatorial plane. Theseeffectsof nccodazolea~ earlier than tlmse of colchicine in the same cells. F) We have studied 17 ~ophases in so-treated eggs, in 15, centrosphereswere r-&visiblewitha pMxm.icroscopeard 2 did shw small blocked centrospheres. 2°/Effectsof a 45minutetrea~tbeginnirq 45mjnutesafterto (suppossa eixneof'~~se), Lln~ofdeL?to Stlid~ the effects oFmcodazole at'tekghase (rmmally about 9Ominutes). Thecentrosphereswerea littledenser ardmeheavily stained thannormally. Sane,astral fibreswarepresent, butfwer ard tshrter than rormally (fig. 3, 6, 7, 8). Then these centrospheseswerenotcanpletelyblocked. There canbe eit&r a single
506
Cell Biology
International
Reports,
Vol. 1, No. 6, 19 77
transversally lenghtened centrosphere (fig. 3, 6) or TV centrospheres (fig. 7, 8) more or less separated one from the other, according to thetimewhentheblcckingcccurred.. Whenthe treated eggs containedmetaphases or anaphases, the centrosphereswereblocked (9mitoses) or absent (2mitoses).When they contained telophase, 4 mitoses were blocked and 23 only partially. 3’/
Effects of a 45 minute treatment beginning at to with ensuing recoveryduring 9ominutes.
A) The nuclei, free of centrospheres and fibres (apolar nuclei), were not in the central region of the blastaneres, but nearer tothecortexard theanimalpole, always embedded inanon vitelline cytoplasn inthe formof a cupola, as if they had carried with then the surrourding hyaloplasm. The process of depolarization continusd innormalmedium. B) The structure of nuclei was always telophasic (with karyomeres) or telo-pophasic. There were usually tx~ nuclei for an hyaloplasmic zone, in scme cases they had fused, C) In the central part of sane nuclei chratrxanes were not ccqletely transformed into chrwsanal vesicles, while this transformation was made at their periphary. Itisnotewxthythat,atthecorrespoxxUngtime,untreated eggswereatthemiddleof thefourthcycle (4metaphases) at the end of the third (2 telophases). 4”/
Effectsof atreatmentduring toardduring 9ominutes.
ard not
the secordmitisisbeginniqat
Thecentrosphereswereccmpletelyblocl&,nrxe~eneXs after 45 minutes, but their structure rsfnaird more or less granular. Scxne"rosette" nuclei appeared (fig. 9). than
5'/ Action of the same concentration stage of segmentation.
during 90 minutes at any
In sane morulas, centered nuclei ("rosettes") could be observed (fig. 9). The centrospheres were ccfrpletely blocked at any phase of mitosis : for instance at mataphase (fig. lo), telophase or telo-wophase (fig. 11) ; they were dense, strongly staindard freeof f&es, scxnetimeswithaless stained center ("enpty centrospheres) (fig. 12). The closer the cycle came to its erd, the smaller the centrospheres.
Cell Biology
International
Reports,
Vol. 1, No. 6, 19 7 7
508
Cell Biology International
Repotts, Vol. 1, No. 6, 1977
lo/ Atmetaphase of the secord mitisis, the sianiltanexs disap pearanceof theastral fibresard of the polar parts of the spindle fibres,whiletheir equatorialpartsare~e~~,mustbe~~inedbyadifference insensitivity of thesets+otypesof fibres, which are of different origin (respectively as-led by the pericentriolar ardbythekinetocboreorganizingcenters). Irde& the former arer&rmallyassembled beforethelatter. Theastralfibres ~thepolarpartsafthespirdlef~esbehaveinthesame~y. Intheabaeof the form&r, theequatiialpartsof the spirdle fibres aaulii mtbeorientior stretched. 2”/ Atmetaphasetherecognisable centrosphereswit.l-outastral fibres, blocked after a 45 minute trealment, always appearmore orlessgrarxUr.This typeof centrospherewasobs~ed at the beginning of the action of saturated qutiline (see SDJI!EIN 1970 fig. 13 a 17, 27 and 46). EUt after a 90 minute treatment, dense, lxmngeneous, srooth-surfacedcentrosphereswerefaund. The former type seems lmbe thefirststepof the latter.
3'/ The fact that the astral fibres do r& canpletely disappear at the end of telophase after 45 minutes, suggests that rxxodazole destsaysthanlessquickly~theastersareexpansivethan whentheybeccme smaller. 4’/ Thecentrospheresarerarelyvisible atwophaseafter action of rrxcdazole. In similar cases, when the cycle may be considered as relatively slowed down, this effect could have begun at the Theabsenzeofpericentriolar preceding anaphaseortelophase. materialcculdbedueeither toaninhibitionof themicrotubule assembly at anaphase or to an enharrzenent of their disassgnbly attelophase.
5'/ The centrospheres are n-ore qletely blocked after.a longer treatment (9Ominutes), lxtcanpletelydisappear, as well as the fibres, 9Ominutesafter the erd of a 45minutetreatment. This ~ndoesnot~thatnxodazolehas inanywayanirre versible action,butprobablyreflectsthe solidbindingof the drug tomicrotubule subunits. 6'/ All the observations made on segmentation mitoses lead to the conclusion that noccdazole has not the same effects as colchicine : it does not make centrospheres disappear, they are "blocked" as with quinoline. The question still staMs : is the difference a qualitative one or is it a cons-e of the rapid action of noccdazoleascanpared toother spindle inhibitors? Fig. 3, 6, 7, 8 -Same treatmtm&telophase. Fig. 9 to 12 - Noccdazole 1 H 30 min.
Ceil Biology International
509
Reports, Vol. 1, NO. 6, 1977
ATES, Y. et SENlXN, P. (1977) Centrospheres ad cellular in the cleaving eggs of Pleur&eles waltlii Michah. Biologiecellulaire, sous Fesse.
cycle
DE BRABANDER, M., VAN DE VElRE, R., AEliTS, P., GZJENSG., axd DE CREE, J. (1975) Orkxdazole EDRZRS, M., DESPm,L. (R. 17934) : anewanticarxzerdrug interferingwithmicrotubules. Effect on neoplastic cells cultured in vitro and 2 vivo.Microtubulesardmicrotubule inhibitors, eds.M. BXGEBS et. DE Bl?AmNDER,509-521. DE BRAIBNDEEt,M., DEMEY,J., SONIAU, M. and GWENS, G. (1977) Ultrastructural ~enical distribution of tubulin incultured cells treatedwithmicrotubule inhibitors.Cell Biology Intern. Reports, L, 177-183. SENTETN,P. (1965) Ananalies des tiles obmes par l'action du sel&ium sur les mitoses de segmentation chez des es&&es r&sistantes. Canparaison avec l'effet de l'&hylamino%sac&ylcolchicide. C. R. Acad. Sci. Paris, 260, 5904-5906. Sm, P. (1970) Action de la quinoline sur les mitoses de segmentation des oeufs d'Urod~les : le blocage de la centrosphere. Chrmsc~~ (B-l.), 32, 97-134. S-IN, P. (1976) Methods of fixing, sectioning ard staining Amphibianeggs for cytological study.MicroscopicaActa,7& 427-438.
SENPEIN, P. et ATES, Y. (1974) Action de l'hydrate de chloral sur les mitoses de segmentation de l'oeuf de Pleurcdele. Etude cytologique et ultrastructurale. Chratosana (Berl.), 45, 215-244.