- Email: [email protected]
Intranuclear microtubules in Tetrahymena pyriformis GL
Experimenlol
Cell Research
53, 8.j-.93
(1968)
INTRANUCLEAR
MICROTUBULES
TETRAHYMENA J. ITO,l Deportment
IN
PYRIFORMIS
Y. C. LEE2
GL
and 0. H. SCHERBAUM2
of‘ Electron Microscopy, City of Hope Medical Center,l Duarte, Calif. and Department of Zoology, University of Cnlifornia, Los Angeles, Cnlif. 90024, USA Received
February
91010,
29, 1968
“microtubules” were demonstrated by the use of glutarcytoplasmic aldehyde-osmium double fixation [7, 181, their ubiquity has been proven in a wide variety of animal and plant cells [ 1, 5, 6, 9, 11, l-5, 21l25]. HOW ever, in spite of general agreement as to the presence and morphology of the microtubules, their function in resting cells is still poorly understood. The occurrence of intranuclear microtubules has been reported in many protozoa [3, 4, 12, 14, 24, 251 and in spermatocytes of the fly [2]. These microtubular structures in nuclei seem to occur only during division of the nuclei. The majority of observers are reasonably inclined to correlate the intranuclear microtubules \\-ith nuclear division, particularly in mitotic division. They have also been found in nondividing insect epidermal cells [19], in which they showed a crystalline array. Tetrahymena pyriforn7is GI,, an amicronucleate strain of ciliated protozoan, was found to have intranuclcar microtubules as a persistent component of the macronucleus throughout its life cycle. This report will describe the structure and disposition of the intranuclear microtubules in the Tetru17 yme17c1 cells, both normal and heat-treated for synchronous division, and will discuss their possible role in the macronuclear events. SIR.~E
MATERIALS
AND
METHODS
Tetrahymena pyriformis GL was grown aseptically in a proteose-peptone medium, using 2 per cent proteose-peptone, 0.1 per cent potassium phosphate, 0.16 per cent sodium acetate hydrous and 0.1 per cent Bacto-dextrose. The heat-synchronization 1 Supported * Supported D.C. This work USA.
by City in part
of Hope NIH General Research Support by contract NONR-233-71 from Office
was completed
al St Jude
Children’s
Research
Award. of Naval Hospital,
Research,
Washington,
.\Iemphis,
Term.
Experimental
38101,
Cell Research
53
86
J. Ito,
Y. C. Lee and 0. H. Scherbaum
treatment (HT), consisting of seven intermittent heat shocks of 30-min intervals, was started with a population of 50 x lo3 cells/ml [IS, 171. Ten ml aliquots of the cells were sampled at various times from an 8 1 mass culture. Samples were taken during the normal log phase immediately prior to HT; during HT and the recovery period; at the 1st synchronous division; shortly after the 1st division; in the maximum stationary phase in which the cell population was 200 x103/ml. Cells were double-fixed with 6.25 per cent glutaraldehyde in 0.1 121 phosphate buffer followed by 1 per cent 0~0, in the Millonig’s buffer, at pHs 7.2, and embedded in an epoxy resin mixture [20]. Ultrathin sections were double-stained with saturated uranyl acetate and Reynolds’ lead citrate 1101 before examination with an electron microscope. RESULTS It is well knolvn that the macronucleus of T. pyriformis has t\\-o clifl’erent types of granules, chromatin hotiies anti nucleoli. In the present study, hesides these nuclear components, a variable number of rather long and straight microtuhules were generally found in the macronuclei of the Tetrcrhymrnrr cells in all stages examined. The outer diameter of these intranuclear microtubules mcasurccl 230 to 300 a. The full length was not determined, hut some microtubules were up to 3 ,u in length (Fig. 3). In longitudinal sections the lumen of the microtubules appearetl slightly denser than the nucleoplasmic hackgroun(1 (Fig. 1). Often, two to seven and sometimes more microtubules \\-ere arrangetl in tightly packed bundles (Figs 1, 2, 4 ant1 7). The bundles of microtubules seemecl to he irregularly coated \v;th strongly osmiophilic materials. This osmiophilic substance appeared to be finely granular anti similar in composition to chromatin hotlies and nucleoli (Figs 1, 2 ant1 4). The adhering granular substance was so massively attached in places that it appeareti dense anti formetl larger granules that \\-ere hartlly distinguishable from the ralhcl small chromatin hodies (Fig. 4). In contrast to the groupecl microtuhules, the single microtubules appeareti to he free of this granular coat (Figs .i and 6). Though the intranuclear microtuhules \vere frequently seen in pro?;imity to the c,hromatin bodies, to the nucleoli and also to the nuclear envelope (Figs 1~ 3 and 5 -7) their tlefinite relation to any specific nuclear components \\-as not clearly demonstrated. The microtubules lying near the nuclear erlrelope were frequently ohservcd in the protruded portion of the marronucleus at nondividing stages, ant1 in the constriction or separation area of the macronucleus at division (Figs 5 and 7). The microtubules were evidently one of the persistent macronuclear components of T. pyriformis GL. They were invariably discernible (luring all Experimental
Cell Research
53
Intranuclear
microtubules
87
phases of cell cycle and did not seem to hare any visible association with the macronuclear events occurring in the synchronous cell division. In the cells sampled at the end of HT, the chromatin bodies \vere aggregated in the middle of the macronuclei, apart from the nuclear envelope (Fig. 3). This aggregation was only observed in the cells sampled immediately after the end of HT and was no longer apparent in the cells sampled 30 min after the end of HT. The intranuclear microtubules seemed to become more prominent at the time of the aggregation of chromatin bodies (Fig. 3). A small number of microtubules, identical to the intranuclear ones, \yere also observed in the perinuclear area of the cytoplasm near the macronuclear envelope (Fig. 5). These cptoplasmic perinuclear microtubules jvere usually short in length and could not be proven to be continuous, either \vith the oriented elements of cilia or with the microtubules running underneath the ectoplasmic layer. No morphological association of the cytoplasmic microtubules with other cytoplasmic components or with the macronuclear envelope \vas seen. DISCUSSION
Spindle fibrils of micronuclei which divide by mitosis, have long been known in micronucleated T. pyriformis and in some other ciliates [14]. The presence of fibers within the macronuclei of ciliates has been described previously, although the fibers were not demonstrated to be tubular. Roth and Minick [13] described the formation of macronuclear fibrillar elements in the late stages of fission of 7’. pyriformis HAJI3. They also reported that these elements tend lo form near the constriction of the nucleus, even though they do not perform a mitotic function in this micronucleate strain. In the amicronucleate strain 7’. pyriformis GL, ho\vever, the intranuclear microtubules lvere a persistent nuclear component. They A\-ere observed throughout all stages examined, and I\-ere found near the chromatin bodies, nucleoli and to the macronuclear envelope. T. pyriformis GL is an amicronucleate strain and divides by fission in which the macronuclei are involved. Therefore, a mitotic apparatus \\-ould not be necessary in this amicronucleate strain. ,\Iicrotubules might simply bc a remnant of the mitotic apparatus which in this strain is no longer functional, but presumably played a role in mitotic division of the micronucleated strain. In contrast to the non-treated T. pyriformis W, in which chromatin bodyaggregation occurred in the early stage of macronuclear division [13I, the aggregation of chromatin bodies appeared to occur much earlier in the heatsynchronized T. pyriformis GL, about 80 min prior to the onset of the 1st
88
I’ig.
J.
1.-A
(no), which GL at log Fig.
tightly
are phase
2.-Transverse closely
substar~ce
Experimenlal
Cell
packed normally immediately sections associated Research
Ito,
bundle distributed prior
Y. C. Lee
0.
of intranuclcar at the periphery to heat-treatment.
of intranuclear with cbromatin 53
and
H.
microtubulcs (nmt) of III~CI.UIIUC~~US. x 40,000.
microtubules hodies
Scherbnrlm
(nmt) (13).
x 96,000.
that
running close Tefrulryrr~erfu are
embedded
to nucleoli p~riformis in
a granular
Intranuclear
Fig. 3.--7’. pqriformis bodies (cb) within indicate a microtubule ning near a nucleolus. m, mitochondrion;
microtubules
89
GL al Lhe end of heat-treatment showing central aggregation of chromatin macronucleus. Nucleoli (no) remain at the periphery. The left upper arrows measuring approx. 3 p. The rig111 lower arrow shows a lnicrolubulc runnmt, Intranuclear microtubulcs; no, nucleoli; IXF, macronuclear envelope; gf, glycogen field. x 8700. Erperimenlrtl
Cell
Research
53
Fig. 4.--h bundle of intranaclear microtubules (runt) embedded in an adhering granular sul~stance At onr point (rrrrom~) this granular srlhstancr is hardly distinguishahlr from a rhromatin hod) (cb). I’. pyriformis GL at the end of beat-lreament. IZD, Nucleolus; ne, macronuclear envelope. x 46,000. Fig. 5.--Syncluxu~ously dwiding 2’. ~qri/ormis GL showing microlubules wilbin a ~nacr~nuclear protrusion (arrow). Cytoplasmic microtubules (cmt) are also found in the peri-nuclear arca. no, Elongated nucleolus; cb, chromatin bodies; TX, macronuclcar envelope; er, rough-surfaced endoplasmic reticulum; m, mitochondrion. x lN,OOO. Fig. 6.--An x 41 .lJoo.
intranuclear
microtubule
running
close
to a nucleolus
(no)
in
a stationary
phase
cell.
Intranuclear
microtubules
9I
Fig. 7.--Syncl1ronously dwiding Y‘. pyriformis GL showing bundles of microtubules within separation areas of 2 daughter nuclei (arrows). The insert shows a higher magnification of the bundled inlranuclear microtubules at the lowrr auww. nmt, Intranuclear microtubules: cb, chromatin hodies, no, nrwlcoli; iu, food vacuole; rn, milochondria; mc, mucocyte. 15,600 and v 27,000. Resenrch
53
J. Ito, Y. C. Lee and 0. H. Scherbaum
92
synchronous division, and did not appear to remain until the synchronous division set in. At the time of chromatin body-aggregation, the intranuclear microtubules appeared to become more discernible. It \vas not determined whether the absolute numbers of the microtubules increased, or whether they remained unchanged. They, however, tended to be more prominent in the nuclear protrusion, and constriction or separation areas. These observations, together with their disposition in the macronuclei, might suggest a possible role in macronuclear division e.g., as a connecting link or attachment of chromatin bodies and nucleoli with the macronuclear envelope. This mechanism might assist the polyploid macronuclei to maintain the individuality of diploid subunits hvhich are postulated to occur during macronuclear tlivision [8]. As suggested for cytoplasmic microtubules in other cells [18], these intranuclear microtubules might also serve as a transport system for cell metabolites between the nuclei and cytoplasm in combination with the perinuclear cgtoplasmic microtubules. SUMMARY hlicrotubules with a diameter of 250-300 A kvere found in the macronuclei of Tetrahymencr pyriformis GL, both normal and heat-synchronized cells, throughout the cell cycle. These microtubules were found to be a persistent macronuclear component. Their possible functions were discussed. The authors are indebted to Drs L. J. Journey and W. W. Johnson, St Jude Children’s Research Hospital, for valuable advice and discussion.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
BEHNKE, O., J. UZfrasfrucf. Res. 11, 139 (1964). BEHNKE, O., and FORER, A., Science 153,1536 (1966). BLUM, J. J., SOMMER, J. R. and KAH, V., J. Profozool. 12, 202 (1965). C~nasso, N. and FAVARD, P., .I. Microscopic 4, 395 (1965). HAYDON; G. B. and TAYLOR, D. A., J. Ceil Biol. 26, 673 (1965). JOURXEY, L. J., Cancer Res. 24, 1391 (1964). LEDBETTER, M. C. and PORTER, K. R., J. Cell Riol. 19, 239 (1963). NANNEY, D. L. and RUDZINSKA, M. A., in J. BRACHET, and A. E. MIRSKY (eds), The Vol. 4, p. 109. Academic Press, New York, 1960. PITELKA, D. R., Electron Microscopic Structure of Protozoa, p. 236. Pergamon Press, York. 1963. REYNOLDS: E. S., J. Cell Biol. 17, 208 (1963). ROBINOW, C. F. and MARAK, J., .I. Cell Biol. 29, 129 (1966). ROTH, L. E. and DANIELS, E. W., J. Cell Biol. 12, 56 (1962) ROTH, L. E. and MINICK, 0. T., J. Profozool. 8, 12 (1961).
Experimental
CeZZ Research
53
Cell, Sew
Intranuclear
microtubules
ROTH, L. E. and SHIGENAKA, Y., J. Cell Hiol. 20, 249 (1964). SODBORN, E., DOEK, P. Ii., MCNABB, J. D. and MOORE, G., (1964). 16. SCHERBAUM, 0. and ZEUTHEN, E., Exptl Cell Res. 6, 221 (1954). Ii. ~ Ibid. Suppl. 3, 312 (1955). 18. SLAUTTERBACK, D. B., J. Cell Biol. 18, 36T (1963). 19. SMITII, U. and SMITH, D. S., J. Cell Bio!. 26, 961 (1965). 20. SITR, A. R., in So. Calif. Sot. Electron Microscopy, 19th Anniv. California, Los Angeles, L. A., Calif. 90024, ITSA. 21. TAYLOR, A. C., .I. Cell Biol. 28, 155 (1966). 22. TX&., G. DE., .I. Cell Biol. 23, 265 (1964). 23. TILNEY, I,. G. and PORTER, K. R., Protoptasma 60, 21 (1965). 21. \‘IVIER, E., J. Microscopic 4, 559 (1965). 2.5. \YISE, B. S., J. Cell Rio!. 27, 113X (1965). 14. 15.
93 J.
C’/trastruct.
Meeting,
Experimental
Res.
Dec.,
1961,
11,
123,
IJniv.
Cell Research
53
Cell Research
53, 8.j-.93
(1968)
INTRANUCLEAR
MICROTUBULES
TETRAHYMENA J. ITO,l Deportment
IN
PYRIFORMIS
Y. C. LEE2
GL
and 0. H. SCHERBAUM2
of‘ Electron Microscopy, City of Hope Medical Center,l Duarte, Calif. and Department of Zoology, University of Cnlifornia, Los Angeles, Cnlif. 90024, USA Received
February
91010,
29, 1968
“microtubules” were demonstrated by the use of glutarcytoplasmic aldehyde-osmium double fixation [7, 181, their ubiquity has been proven in a wide variety of animal and plant cells [ 1, 5, 6, 9, 11, l-5, 21l25]. HOW ever, in spite of general agreement as to the presence and morphology of the microtubules, their function in resting cells is still poorly understood. The occurrence of intranuclear microtubules has been reported in many protozoa [3, 4, 12, 14, 24, 251 and in spermatocytes of the fly [2]. These microtubular structures in nuclei seem to occur only during division of the nuclei. The majority of observers are reasonably inclined to correlate the intranuclear microtubules \\-ith nuclear division, particularly in mitotic division. They have also been found in nondividing insect epidermal cells [19], in which they showed a crystalline array. Tetrahymena pyriforn7is GI,, an amicronucleate strain of ciliated protozoan, was found to have intranuclcar microtubules as a persistent component of the macronucleus throughout its life cycle. This report will describe the structure and disposition of the intranuclear microtubules in the Tetru17 yme17c1 cells, both normal and heat-treated for synchronous division, and will discuss their possible role in the macronuclear events. SIR.~E
MATERIALS
AND
METHODS
Tetrahymena pyriformis GL was grown aseptically in a proteose-peptone medium, using 2 per cent proteose-peptone, 0.1 per cent potassium phosphate, 0.16 per cent sodium acetate hydrous and 0.1 per cent Bacto-dextrose. The heat-synchronization 1 Supported * Supported D.C. This work USA.
by City in part
of Hope NIH General Research Support by contract NONR-233-71 from Office
was completed
al St Jude
Children’s
Research
Award. of Naval Hospital,
Research,
Washington,
.\Iemphis,
Term.
Experimental
38101,
Cell Research
53
86
J. Ito,
Y. C. Lee and 0. H. Scherbaum
treatment (HT), consisting of seven intermittent heat shocks of 30-min intervals, was started with a population of 50 x lo3 cells/ml [IS, 171. Ten ml aliquots of the cells were sampled at various times from an 8 1 mass culture. Samples were taken during the normal log phase immediately prior to HT; during HT and the recovery period; at the 1st synchronous division; shortly after the 1st division; in the maximum stationary phase in which the cell population was 200 x103/ml. Cells were double-fixed with 6.25 per cent glutaraldehyde in 0.1 121 phosphate buffer followed by 1 per cent 0~0, in the Millonig’s buffer, at pHs 7.2, and embedded in an epoxy resin mixture [20]. Ultrathin sections were double-stained with saturated uranyl acetate and Reynolds’ lead citrate 1101 before examination with an electron microscope. RESULTS It is well knolvn that the macronucleus of T. pyriformis has t\\-o clifl’erent types of granules, chromatin hotiies anti nucleoli. In the present study, hesides these nuclear components, a variable number of rather long and straight microtuhules were generally found in the macronuclei of the Tetrcrhymrnrr cells in all stages examined. The outer diameter of these intranuclear microtubules mcasurccl 230 to 300 a. The full length was not determined, hut some microtubules were up to 3 ,u in length (Fig. 3). In longitudinal sections the lumen of the microtubules appearetl slightly denser than the nucleoplasmic hackgroun(1 (Fig. 1). Often, two to seven and sometimes more microtubules \\-ere arrangetl in tightly packed bundles (Figs 1, 2, 4 ant1 7). The bundles of microtubules seemecl to he irregularly coated \v;th strongly osmiophilic materials. This osmiophilic substance appeared to be finely granular anti similar in composition to chromatin hotlies and nucleoli (Figs 1, 2 ant1 4). The adhering granular substance was so massively attached in places that it appeareti dense anti formetl larger granules that \\-ere hartlly distinguishable from the ralhcl small chromatin hodies (Fig. 4). In contrast to the groupecl microtuhules, the single microtubules appeareti to he free of this granular coat (Figs .i and 6). Though the intranuclear microtuhules \vere frequently seen in pro?;imity to the c,hromatin bodies, to the nucleoli and also to the nuclear envelope (Figs 1~ 3 and 5 -7) their tlefinite relation to any specific nuclear components \\-as not clearly demonstrated. The microtubules lying near the nuclear erlrelope were frequently ohservcd in the protruded portion of the marronucleus at nondividing stages, ant1 in the constriction or separation area of the macronucleus at division (Figs 5 and 7). The microtubules were evidently one of the persistent macronuclear components of T. pyriformis GL. They were invariably discernible (luring all Experimental
Cell Research
53
Intranuclear
microtubules
87
phases of cell cycle and did not seem to hare any visible association with the macronuclear events occurring in the synchronous cell division. In the cells sampled at the end of HT, the chromatin bodies \vere aggregated in the middle of the macronuclei, apart from the nuclear envelope (Fig. 3). This aggregation was only observed in the cells sampled immediately after the end of HT and was no longer apparent in the cells sampled 30 min after the end of HT. The intranuclear microtubules seemed to become more prominent at the time of the aggregation of chromatin bodies (Fig. 3). A small number of microtubules, identical to the intranuclear ones, \yere also observed in the perinuclear area of the cytoplasm near the macronuclear envelope (Fig. 5). These cptoplasmic perinuclear microtubules jvere usually short in length and could not be proven to be continuous, either \vith the oriented elements of cilia or with the microtubules running underneath the ectoplasmic layer. No morphological association of the cytoplasmic microtubules with other cytoplasmic components or with the macronuclear envelope \vas seen. DISCUSSION
Spindle fibrils of micronuclei which divide by mitosis, have long been known in micronucleated T. pyriformis and in some other ciliates [14]. The presence of fibers within the macronuclei of ciliates has been described previously, although the fibers were not demonstrated to be tubular. Roth and Minick [13] described the formation of macronuclear fibrillar elements in the late stages of fission of 7’. pyriformis HAJI3. They also reported that these elements tend lo form near the constriction of the nucleus, even though they do not perform a mitotic function in this micronucleate strain. In the amicronucleate strain 7’. pyriformis GL, ho\vever, the intranuclear microtubules lvere a persistent nuclear component. They A\-ere observed throughout all stages examined, and I\-ere found near the chromatin bodies, nucleoli and to the macronuclear envelope. T. pyriformis GL is an amicronucleate strain and divides by fission in which the macronuclei are involved. Therefore, a mitotic apparatus \\-ould not be necessary in this amicronucleate strain. ,\Iicrotubules might simply bc a remnant of the mitotic apparatus which in this strain is no longer functional, but presumably played a role in mitotic division of the micronucleated strain. In contrast to the non-treated T. pyriformis W, in which chromatin bodyaggregation occurred in the early stage of macronuclear division [13I, the aggregation of chromatin bodies appeared to occur much earlier in the heatsynchronized T. pyriformis GL, about 80 min prior to the onset of the 1st
88
I’ig.
J.
1.-A
(no), which GL at log Fig.
tightly
are phase
2.-Transverse closely
substar~ce
Experimenlal
Cell
packed normally immediately sections associated Research
Ito,
bundle distributed prior
Y. C. Lee
0.
of intranuclcar at the periphery to heat-treatment.
of intranuclear with cbromatin 53
and
H.
microtubulcs (nmt) of III~CI.UIIUC~~US. x 40,000.
microtubules hodies
Scherbnrlm
(nmt) (13).
x 96,000.
that
running close Tefrulryrr~erfu are
embedded
to nucleoli p~riformis in
a granular
Intranuclear
Fig. 3.--7’. pqriformis bodies (cb) within indicate a microtubule ning near a nucleolus. m, mitochondrion;
microtubules
89
GL al Lhe end of heat-treatment showing central aggregation of chromatin macronucleus. Nucleoli (no) remain at the periphery. The left upper arrows measuring approx. 3 p. The rig111 lower arrow shows a lnicrolubulc runnmt, Intranuclear microtubulcs; no, nucleoli; IXF, macronuclear envelope; gf, glycogen field. x 8700. Erperimenlrtl
Cell
Research
53
Fig. 4.--h bundle of intranaclear microtubules (runt) embedded in an adhering granular sul~stance At onr point (rrrrom~) this granular srlhstancr is hardly distinguishahlr from a rhromatin hod) (cb). I’. pyriformis GL at the end of beat-lreament. IZD, Nucleolus; ne, macronuclear envelope. x 46,000. Fig. 5.--Syncluxu~ously dwiding 2’. ~qri/ormis GL showing microlubules wilbin a ~nacr~nuclear protrusion (arrow). Cytoplasmic microtubules (cmt) are also found in the peri-nuclear arca. no, Elongated nucleolus; cb, chromatin bodies; TX, macronuclcar envelope; er, rough-surfaced endoplasmic reticulum; m, mitochondrion. x lN,OOO. Fig. 6.--An x 41 .lJoo.
intranuclear
microtubule
running
close
to a nucleolus
(no)
in
a stationary
phase
cell.
Intranuclear
microtubules
9I
Fig. 7.--Syncl1ronously dwiding Y‘. pyriformis GL showing bundles of microtubules within separation areas of 2 daughter nuclei (arrows). The insert shows a higher magnification of the bundled inlranuclear microtubules at the lowrr auww. nmt, Intranuclear microtubules: cb, chromatin hodies, no, nrwlcoli; iu, food vacuole; rn, milochondria; mc, mucocyte. 15,600 and v 27,000. Resenrch
53
J. Ito, Y. C. Lee and 0. H. Scherbaum
92
synchronous division, and did not appear to remain until the synchronous division set in. At the time of chromatin body-aggregation, the intranuclear microtubules appeared to become more discernible. It \vas not determined whether the absolute numbers of the microtubules increased, or whether they remained unchanged. They, however, tended to be more prominent in the nuclear protrusion, and constriction or separation areas. These observations, together with their disposition in the macronuclei, might suggest a possible role in macronuclear division e.g., as a connecting link or attachment of chromatin bodies and nucleoli with the macronuclear envelope. This mechanism might assist the polyploid macronuclei to maintain the individuality of diploid subunits hvhich are postulated to occur during macronuclear tlivision [8]. As suggested for cytoplasmic microtubules in other cells [18], these intranuclear microtubules might also serve as a transport system for cell metabolites between the nuclei and cytoplasm in combination with the perinuclear cgtoplasmic microtubules. SUMMARY hlicrotubules with a diameter of 250-300 A kvere found in the macronuclei of Tetrahymencr pyriformis GL, both normal and heat-synchronized cells, throughout the cell cycle. These microtubules were found to be a persistent macronuclear component. Their possible functions were discussed. The authors are indebted to Drs L. J. Journey and W. W. Johnson, St Jude Children’s Research Hospital, for valuable advice and discussion.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
BEHNKE, O., J. UZfrasfrucf. Res. 11, 139 (1964). BEHNKE, O., and FORER, A., Science 153,1536 (1966). BLUM, J. J., SOMMER, J. R. and KAH, V., J. Profozool. 12, 202 (1965). C~nasso, N. and FAVARD, P., .I. Microscopic 4, 395 (1965). HAYDON; G. B. and TAYLOR, D. A., J. Ceil Biol. 26, 673 (1965). JOURXEY, L. J., Cancer Res. 24, 1391 (1964). LEDBETTER, M. C. and PORTER, K. R., J. Cell Riol. 19, 239 (1963). NANNEY, D. L. and RUDZINSKA, M. A., in J. BRACHET, and A. E. MIRSKY (eds), The Vol. 4, p. 109. Academic Press, New York, 1960. PITELKA, D. R., Electron Microscopic Structure of Protozoa, p. 236. Pergamon Press, York. 1963. REYNOLDS: E. S., J. Cell Biol. 17, 208 (1963). ROBINOW, C. F. and MARAK, J., .I. Cell Biol. 29, 129 (1966). ROTH, L. E. and DANIELS, E. W., J. Cell Biol. 12, 56 (1962) ROTH, L. E. and MINICK, 0. T., J. Profozool. 8, 12 (1961).
Experimental
CeZZ Research
53
Cell, Sew
Intranuclear
microtubules
ROTH, L. E. and SHIGENAKA, Y., J. Cell Hiol. 20, 249 (1964). SODBORN, E., DOEK, P. Ii., MCNABB, J. D. and MOORE, G., (1964). 16. SCHERBAUM, 0. and ZEUTHEN, E., Exptl Cell Res. 6, 221 (1954). Ii. ~ Ibid. Suppl. 3, 312 (1955). 18. SLAUTTERBACK, D. B., J. Cell Biol. 18, 36T (1963). 19. SMITII, U. and SMITH, D. S., J. Cell Bio!. 26, 961 (1965). 20. SITR, A. R., in So. Calif. Sot. Electron Microscopy, 19th Anniv. California, Los Angeles, L. A., Calif. 90024, ITSA. 21. TAYLOR, A. C., .I. Cell Biol. 28, 155 (1966). 22. TX&., G. DE., .I. Cell Biol. 23, 265 (1964). 23. TILNEY, I,. G. and PORTER, K. R., Protoptasma 60, 21 (1965). 21. \‘IVIER, E., J. Microscopic 4, 559 (1965). 2.5. \YISE, B. S., J. Cell Rio!. 27, 113X (1965). 14. 15.
93 J.
C’/trastruct.
Meeting,
Experimental
Res.
Dec.,
1961,
11,
123,
IJniv.
Cell Research
53