EXPERIMENTAL
PARASITOI
Entamoeba
OGY
73,
276284 (1991)
histolytica:
Microtubule
FRANCISCO J. SOLE’ Unidad de Microscopia
Movement
during
Mitosis
AND ROBERTO BARRIOS
Electronica, Facultad de Medicina, Mexico, D.F., 04510, Mexico
UNAM,
Circuito
Interior,
SOLIS, F. J., AND BARRIOS, R. (1991). Entamoeba histolytica: Microtubule movement during mitosis. Experimental Parusitology 73, 276-284. The movement of microtubules (MTs) during nuclear division of Entamoeba histolytica was ultrastructurally studied. Regarding this MT movement, five stages of mitosis could be defined: prophase, metaphase, anaphase A, anaphase B, and telophase. In early stages of mitosis, chromatinic material appeared condensed, and MTs were detected in the center of the nucleus. Later, MTs seemed to grow from an electron-dense body located in the center of the nucleus. This body might be the microtubule organizing center, which organized the MTs, first in a lateral way, and later to form the mitotic spindle, which was made of a bundle of MTs joined by their ends. This junction of MTs to themselves could also be observed in cross-sections. The last stage of mitosis was the nuclear separation. Two different morphological types of intranuclear vesicles were also observed, which seemed to have different types of membrane. Both intranuclear vesicles were present during nuclear division, generally in clusters, and located 0 1991 Academic PXSS, h. close to the nuclear periphery. INDEX DESCRIPTORS AND ABBREVIATIONS: Entamoeba histolytica; Microtubules; Mitosis; Microtubule organizing center; Intr-anuclear vesicles; Microtubules (MTs); Microtubule organizing center (MTOC); lntranuclear vesicles (IV).
The movement of MTs during mitosis has been widely described in relation with the traditional subdivisions of the mitotic cycle, from prophase to telophase (Dustin 1982). In prophase and metaphase stages, some cells, such as mammalian cells, bring the centrioles to the poles of the nucleus and the chromosomes become apparent. The MTs assemble the two halves of the mitotic spindle. In the anaphase stage the chromosomes move to the poles, and the MTs that link the kinetochores (k) to the centrosomal region decrease in length, whereas the MTs that are not attached to the chromosomes often elongate. The polar MTs usually interdigitate in the nuclear equatorial region, and they can be seen as a more electron-dense zone. Finally, in telophase, whereas the daughter nuclei resume their interphasic pattern of chromatin, the k-MTs and the asters vanish progressively, ’ To whom correspondence
should be addressed. 276
0014-4894/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
and the polar MTs persist in bundles which closely assemble into a very elongated structure, showing, at its middle, interdigitated MTs embedded in a dense material: the telophasic bundle, and its midbody. In Entamoeba histofytica, four stages have been described in its nuclear division, according to the nuclear shape: prophaselike, early and late (or B), anaphase-like, and telophase-like stages (Solis et al. 1986; Orozco et al. 1988). The sequence of these stages was determined by light and electron microscopy, as well as by autoradiographic studies. However, the lack of ultrastructural details concerning the MTs movement during the nuclear division did not allow us to compare the resemblance of the MTs movement in amoeba to that of mammalian MTs movement in mitosis. To describe the movement of MTs throughout mitosis, we studied ultrastructurally the amoebic nucleus during the different stages of this process. In this paper we describe the MTs move-
E. histolytica: MICROTUBIAL ment during nuclear division in E. histolytica and give new information about a new type of amoebic intranuclear vesicle.
MOVEMENT
277
phasic peripheral material, and it was also arranged regularly around the nucleus, located between the nuclear envelope (NE) and the central electron dense bodies. MATERIALS AND METHODS Some MTs reached the zone where this peCulture of trophozoites. Trophozoites of E. histolyripheric material was located, and were not tica (HMl:IMSS strain and clone) were cultured axe- long enough to reach the nuclear envelope. nically with TYI-S-33 medium (Diamond et al. 1978) Two different types of IVs can be observed and harvested during logarithmic growth phase. Cloning of trophozoites was performed in TYI-S-33 me- close to the nuclear membrane: Doublemembrane IVs and one-membrane IVs (Fig. dium containing 0.5% agar, as reported elsewhere (Gillin and Diamond 1978). 1, arrow; Fig. 3). Electron microscopy studies. The clone isolated As the nuclear division proceeded to the from HMl:IMSS was not synchronized. The nuclear anaphase A stage, a higher level of MTs shape was used as a reference to know the stage of organization was observed as they started mitosis (Orozco et al. 1988). The sample preparation to form a mitotic spindle (Fig. 2). Some for electron microscopy was as follows: the culture medium was removed from the culture flasks containcross-sectioned MTs appeared in the place ing E. histolytica trophozoites at logarithmic growth where the MTs seemed to originate (Fig. 2, phase. Fixation was performed by 2.5% glutaraldedashed arrows). Most of the MTs were hyde in 0.1 M cacodylate buffer for 1 hr. Trophozoites close to this place and seemed to be part of were released from the bottom of the culture flasks by a mitotic spindle, as inferred from their scraping with a rubber policeman. Cells were then postfixed with 1% 0~0, in 0.1 M cacodylate buffer, clustering (Fig. 2, asterisk). Some MTs followed by dehydration using graded series of ethanol were very close to the nuclear membrane and propylene oxide treatments, and then embedded and far from the place of MTs origin (Fig. 2, in Epon 812. Thin sections were obtained and conarrow). There were MTs diverging from trasted with aqueous uranyl acetate and lead citrate, their apparent origin place (Fig. 2, arrowaccording to Reynolds (1%3), and examined with a Zeiss EM-10 electron microscope for nuclear analysis. heads), which were not parallel to the main bundle of MTs (Fig. 2, asterisk). A pair of RESULTS MTs is joined by their ends to another MT About 700 nuclei of E. histolytica tropho- (Fig. 2, circle). Little amounts of conzoites, in logarithmic growth phase, were densed peripheric material could be seen, studied to investigate the MTs movement and most of it was located in the upper part during the nuclear division. The stage of of this nucleus. Another section of amoebic division was determined by the shape of the trophozoites with nuclei in this stage of minuclei according to previous reports (Solis tosis, anaphase A, presented grouped MTs, et al. 1986; Orozco et al. 1988). The size forming a bundle (Fig. 3) that resembles a and shape of amoebic MTs matched the size more organized spindle (S) than the one oband shape of mammalian MTs. Figure 1 served in Fig. 2. The spindle is conformed shows a nucleus in the prophase stage of by short MTs, which seem to be joined by mitosis, which presented many radially dis- their ends. In this section no cross-secposed MTs (MT). They seemed to anchor tioned or lateral MTs were observed. The to small electron-dense bodies, with non- two types of IVs (Fig. 3, V, arrows) were electron-dense zones among them, and lo- present. There are two groups of onecated in the center of the nucleus. Some membrane IVs (Fig. 3, arrows) close to the cross-sectioned MTs were also observed nuclear envelope (NE). The already formed mitotic spindle (S) (Fig. 1, arrowheads). These electron-dense eventually grew, in anaphase B stage of mibodies might correspond to E. histolytica MTOC (M). Some of the peripheric mate- tosis, and extended along the nucleus, rial (PM) was more compact than the inter- through a large and narrow homogeneous
278
SOLIS AND BARRIOS
FIG. I. Prometaphasic plate of Enramoeba histolyrica. The MTOC (M) is electron-dense, surrounded by some peripheral chromatinic material (PM). The microtubules (MT) are arranged in a radial way. Some cross-sectioned MTs are present (arrowheads). There are two different types of intranuclear vesicles. Some of them have a thin membrane (arrow). NE, Nuclear envelope. Bar = 1 pm.
bundle of MTs (Fig. 4A). These MTs were apparently joined to themselves through their ends (Fig. 4A, inset). No crosssectioned MTs were found, probably indicating that all MTs have already reached the spindle at this stage of nuclear division. One of the spindle ends was close to the nuclear envelope, although the MTs of this spindle end did not reach the nuclear envelope. Some electron-dense material was embedded in MTs of the spindle (Fig. 4A, thin arrow). A cross-section of the mitotic spindle is shown in Fig. 4B; MTs occupy the central part of the nucleus. Most of the MTs were joined by electron-dense bridges
(Fig. 4B, inset, arrows), with no apparent regular arrangement. A group of onemembrane IVs (Fig. 4A, thick arrow) is separated from the other type of IVs. The PM was distributed uniformly in these nuclei (Figs. 4A and 4B). During the last stage of nuclear division of E. histolytica, i.e., in the telophase stage, before complete separation of the daughter nuclei, when both of them are still joined (Fig. 5, arrow), many of the MTs (MT) were still present in both nuclei and they seemed to be separated by the nuclear membrane (Fig. 5). Some nuclei had chromosome-like material (Fig. 5, arrowheads).
E. histolytica: MICROTUBIAL MOVEMENT
279
FIG. 2. Microtubule reorganization in Entamoeba histolytica nucleus. The nucleus is in anaphase A stage. The MTs (arrowheads) are diverging from a common zone (dashed arrows). A MT is close to the nuclear membrane (arrow). There is a zone with a bundle of MTs (asterisk). A pair of MTs are joined to another by their ends (circle). Bar = 1 pm.
The peripheric material (PM) is arranged in discrete small bodies. During our observations, we were not able to see the nuclei joined by a thin thread (Orozco et al. 1988); thus, we could not observe the telophasic bundle MTs. Therefore, the existence of a microtubular midbody, similar to that observed in telophasic mammalian cells (Dustin 1982), remains to be determined. DISCUSSION
In this paper, we present a more detailed image of nuclear division in E. histolytica, especially in regard to the movement of MTs during nuclear division. This proto-
zoan presents a closed, possibly intranuclear pleuromitosis (Orozco et al. 1988). The nuclear shape was taken as a reference to know approximately the stages of nuclear division of this parasite, according to previous reports (Solis et al. 1986; Orozco et al. 1988). The MTs movement at the beginning of amoebic nuclear division resembles the mitosis of the micronucleus in Didinium nasutum (Karadhazan and Raikov 1977), as inferred from the ordering of MTs in a radial way during this stage of nuclear division (Fig. 1). In metaphase, the chromosomes and the MTOC form a metaphasic plate in the same way that has been reported for Laredo strain (Gicquaud 1979),
280
SOLIS AND
BARRIOS
nucleus shows a mitotic spindle FIG. 3. Amoebic nucleus in anaphase A stage. Enfamoeba hisrolytica (S) in formation. There are grouped IVs (V). Two of them show thin surface (arrows). The MTs seem to start from the same place (M). NE, nuclear envelope. Bar = 1 pm.
although this strain is not an E. histolytica amoeba (Bhattacharya et al. 1988, among others). According to the nuclear shape, in ana-
phase A stage, the MTs might start to organize the mitotic spindle and, in this stage, some lateral and cross-sectioned MTs were present (Fig. 2, arrowheads, dashed ar-
E.
histdytica:
MICROTUBIAL
MOVEMENT
FIG. 4. (A) Entamoeba histolytica nucleus in anaphase B stage. Enlarged nucleus with joined MTs (inset) in an enlarged one-piece spindle (S). The nuclear growth might occur from rounded to pointed pole. Two groups of vesicles can be seen. One group of IV has a thin membrane (arrow). Some electron-dense material is embedded in MTs of mitotic spindle (thin arrow). Bar = 2 urn. (B) Crosssectioned MTs of the mitotic spindle. The nucleus was cross-sectioned and shows a narrow zone with MTs. Some of them are joined by electron-dense bridges (inset, arrows). Bar = 1 pm.
281
282
SOLIS AND BARRIOS
FIG. 5. Sectioned mitotic spindle. The MTs (MT) of the Entamoeba hisrolytica mitotic spindle seem to be sectioned. The nuclei are still joined (arrow). One of the daughter nuclei presents chromatinic material (arrowheads). PM, Peripheric material. Bar = 2 pm.
rows). Although there were MTs very close to the membrane, we did not observe the junction of mitotic spindle MTs to the nuclear membrane in this or later stages. The
lack of MTs anchorage to the nuclear membrane has been also observed in Zridia (Cessna, 1978). After this stage, the formation (Fig. 3) and growth (Fig. 4A) of a well-
E. histolytica: MICROTUBIAL defined mitotic spindle could take place during the anaphase B stage of amoebic mitosis. In this stage, the mitotic spindle did not show MTs long enough to reach both poles, and there was not a more electrondense middle zone of interdigitating MTs. The typical mitotic spindle is composed of two halves and the middle zone presents a more electron-dense zone, due to interdigitating MTs, in most of the organisms studied (Dustin 1982; McIntosh and Koone 1989). These features might indicate that the amoebic mitotic spindle is formed by a one-piece mitotic spindle. The presence of bridges between MTs (Fig. 4B, inset, arrows) might indicate the anchoring of the MTs among themselves, as suggested by McIntosh and McDonald (1989) for typical mitotic spindles. The possible mechanism for the formation of the mitotic spindle might be the junction of the MTs by their ends (Figs. 2, circle and Fig. 4A, inset). According to the movement of MTs, the model for nuclear division of E. histolytica that we propose is as follows: (1) In prophase, the condensation of chromatinic material in the periphery of the nucleus takes place, and MTs appear, radially arranged, joined to a central body, which might be the MTOC; (2) in metaphase, the condensed chromatinic material is pulled into the center of nucleus by MTs (Solis et al., in preparation), forming a metaphasic plate; (3) in anaphase A and B, the chromosomes are carried to the nuclear poles by the growth of the mitotic spindle; the presence of electron dense material embedded in mitotic spindle (Fig. 4A, thin arrow) might suggest that the chromosome distribution takes place after the mitotic spindle is complete. (4) The telophase stage, at which the nuclei separate (karyokinesis) and the chromatinic material decondenses. This type of mitosis of E. histofytica has been suggested to be a pleuromitosis (in which the mitotic spindle is bilateral in any of the mitotic stages). Most of the protozoan related to amoeba, such as Acan-
MOVEMENT
283
thamoeba (Sawyer and Griffin 1971), Hartmanella (Page, 1967), and Naegleria (Schuster 1975), undergo open or closed orthomitosis (in which the mitotic spindle is always axial). However, in E. histolytica the formation of the mitotic spindle starts when the MTs, originated from a central body, pull the chromosomes towards the central part of the nucleus. Thus, the mitotic spindle is bilateral at this moment, although with a single MTOC, which might indicate a particular pleuromitosis at the beginning of nuclear division. In regard to E. histolytica intranuclear vesicles, there are reports (Feria-Velasco and Trevino, 1972; Zaman, 1973) describing the double-membrane IV. Our results support these data, and present evidence for the existence of another type of IV. This type of IV had a regular, thin surface, with only one membrane, and it sometimes contained fibrous electron-dense material, which is morphologically similar to the nucleoplasmic material. We observed the increase of both IVs during nuclear division. However, the role of the IVs remains to be determined. ACKNOWLEDGMENTS We thank Dr. Esther Orozco for critical reviewing of this manuscript and Mr. Tomas Cruz and Mario Garcia for photographic work. This work was supported by Consejo National de Ciencia y TecnologiaMexico. REFERENCES BHATTACHARYA, S., BHATTACHARYA, A., AND DIAMOND, L. 1988. Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba. Molecular and Biochemical Parasitology 21, 257-262. C&ANA, D. 1978. La mitose gamogonique chez Iridia lucida (Foraminifera, Lagynidae). Annals Science Natural Zoologie s&r 12, 20, 287-320. DIAMOND, L., HARLOW, D. R., AND CUNNICK, C.
1978. A new medium for the axenic cultivation of Enramoeba histolytica and other Entamoeba. Transactions of Royal Society of Tropical Medicine and Hygiene 74, 431-432. DUSTIN, P. 1982. “Microtubules,” 2nd ed. Springer-
Verlag, New York.
284
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FERIA-VELASCO,A., AND TREVIRO, N. 1972. The ultrastructure of trophozoites of Entamoeba histolytica with particular reference to spherical arrangements of osmiophilic cylindrical bodies. Journal of Protozoology
19, 200-211.
GICQUAUD, C. 1979. Etiide de l’ultrastructure noyau et de la mitose de Entamoeba histolytica. ologie Cellulaire
de Bi-
35, 305-312.
GILLIN, F. AND DIAMOND, L. 1978. Clonal growth of Entamoeba histolytica and other species of Ent25, 539amoeba in agar. Journal of Protozoology 541. KARADHAZAN, B., AND RAIKOV, I. 1977. Ultrastructure of the micronucleus of Didinium nasutum (Ciolophora, Gymnostata) during meiosis. Tsifo/ogiya (Leningrad)
19, 1327-1332.
MCINTOSH,J., AND MCDONALD, K. 1989.The mitotic spindle. Scientific American 26(4), 25-34. MCINTOSH,J., KOONE, M. 1989.Mitosis. Science 246, 622-628. OROZCO,E., SOLIS, F., DOMINGUEZ,J., CHAVEZ, B., AND HERNANDEZ, F. 1988. Eatamoeba histolytica: Cell cycle and nuclear division. Experimental Parasitology 67, 85-95.
PAGE, F. 1967. Taxonomic criteria for limax amoeba,
BARRIOS
with descriptions of 3 new species of Hartmanella and 3 of Vahlkampjia. Journal of Protozoology 14, 499-521. RAIKOV, I. 1982. “The Protozoan Nucleus. Morphology and Evolution,” Vol. 9. Springer, Wien. REYNOLDS,E. 1963.The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal
of Cell Biology
17, 208-212.
SAWYER, T., AND GRIFFIN, J. 1971. Acanthamoeba comandoni and A. astronyxis: Taxonomic characteristics of mitotic nuclei, centrosome and cysts. Journal
of Protozoology
18, 382-388.
SCHUSTER,F. 1975. Ultrastructure of mitosis in the amoeboflagellate Naegleria gruberi. Tissue and Cell Research 7, 1-12. SOLIS, F., CHAVEZ, B., AND OROZCO,E. 1986. Fases de la division nuclear en Entamoeba histolytica. Archives de Investigation 100.
Medica (Mexico)
17(l), 95-
ZAMAN, V. 1973. The intranuclear bodies of Entamoeba.
International
Journal
of Parasitology
3,
243-251. Received 10 December 1990;accepted with revision 25 April 1991
PARASITOI
Entamoeba
OGY
73,
276284 (1991)
histolytica:
Microtubule
FRANCISCO J. SOLE’ Unidad de Microscopia
Movement
during
Mitosis
AND ROBERTO BARRIOS
Electronica, Facultad de Medicina, Mexico, D.F., 04510, Mexico
UNAM,
Circuito
Interior,
SOLIS, F. J., AND BARRIOS, R. (1991). Entamoeba histolytica: Microtubule movement during mitosis. Experimental Parusitology 73, 276-284. The movement of microtubules (MTs) during nuclear division of Entamoeba histolytica was ultrastructurally studied. Regarding this MT movement, five stages of mitosis could be defined: prophase, metaphase, anaphase A, anaphase B, and telophase. In early stages of mitosis, chromatinic material appeared condensed, and MTs were detected in the center of the nucleus. Later, MTs seemed to grow from an electron-dense body located in the center of the nucleus. This body might be the microtubule organizing center, which organized the MTs, first in a lateral way, and later to form the mitotic spindle, which was made of a bundle of MTs joined by their ends. This junction of MTs to themselves could also be observed in cross-sections. The last stage of mitosis was the nuclear separation. Two different morphological types of intranuclear vesicles were also observed, which seemed to have different types of membrane. Both intranuclear vesicles were present during nuclear division, generally in clusters, and located 0 1991 Academic PXSS, h. close to the nuclear periphery. INDEX DESCRIPTORS AND ABBREVIATIONS: Entamoeba histolytica; Microtubules; Mitosis; Microtubule organizing center; Intr-anuclear vesicles; Microtubules (MTs); Microtubule organizing center (MTOC); lntranuclear vesicles (IV).
The movement of MTs during mitosis has been widely described in relation with the traditional subdivisions of the mitotic cycle, from prophase to telophase (Dustin 1982). In prophase and metaphase stages, some cells, such as mammalian cells, bring the centrioles to the poles of the nucleus and the chromosomes become apparent. The MTs assemble the two halves of the mitotic spindle. In the anaphase stage the chromosomes move to the poles, and the MTs that link the kinetochores (k) to the centrosomal region decrease in length, whereas the MTs that are not attached to the chromosomes often elongate. The polar MTs usually interdigitate in the nuclear equatorial region, and they can be seen as a more electron-dense zone. Finally, in telophase, whereas the daughter nuclei resume their interphasic pattern of chromatin, the k-MTs and the asters vanish progressively, ’ To whom correspondence
should be addressed. 276
0014-4894/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
and the polar MTs persist in bundles which closely assemble into a very elongated structure, showing, at its middle, interdigitated MTs embedded in a dense material: the telophasic bundle, and its midbody. In Entamoeba histofytica, four stages have been described in its nuclear division, according to the nuclear shape: prophaselike, early and late (or B), anaphase-like, and telophase-like stages (Solis et al. 1986; Orozco et al. 1988). The sequence of these stages was determined by light and electron microscopy, as well as by autoradiographic studies. However, the lack of ultrastructural details concerning the MTs movement during the nuclear division did not allow us to compare the resemblance of the MTs movement in amoeba to that of mammalian MTs movement in mitosis. To describe the movement of MTs throughout mitosis, we studied ultrastructurally the amoebic nucleus during the different stages of this process. In this paper we describe the MTs move-
E. histolytica: MICROTUBIAL ment during nuclear division in E. histolytica and give new information about a new type of amoebic intranuclear vesicle.
MOVEMENT
277
phasic peripheral material, and it was also arranged regularly around the nucleus, located between the nuclear envelope (NE) and the central electron dense bodies. MATERIALS AND METHODS Some MTs reached the zone where this peCulture of trophozoites. Trophozoites of E. histolyripheric material was located, and were not tica (HMl:IMSS strain and clone) were cultured axe- long enough to reach the nuclear envelope. nically with TYI-S-33 medium (Diamond et al. 1978) Two different types of IVs can be observed and harvested during logarithmic growth phase. Cloning of trophozoites was performed in TYI-S-33 me- close to the nuclear membrane: Doublemembrane IVs and one-membrane IVs (Fig. dium containing 0.5% agar, as reported elsewhere (Gillin and Diamond 1978). 1, arrow; Fig. 3). Electron microscopy studies. The clone isolated As the nuclear division proceeded to the from HMl:IMSS was not synchronized. The nuclear anaphase A stage, a higher level of MTs shape was used as a reference to know the stage of organization was observed as they started mitosis (Orozco et al. 1988). The sample preparation to form a mitotic spindle (Fig. 2). Some for electron microscopy was as follows: the culture medium was removed from the culture flasks containcross-sectioned MTs appeared in the place ing E. histolytica trophozoites at logarithmic growth where the MTs seemed to originate (Fig. 2, phase. Fixation was performed by 2.5% glutaraldedashed arrows). Most of the MTs were hyde in 0.1 M cacodylate buffer for 1 hr. Trophozoites close to this place and seemed to be part of were released from the bottom of the culture flasks by a mitotic spindle, as inferred from their scraping with a rubber policeman. Cells were then postfixed with 1% 0~0, in 0.1 M cacodylate buffer, clustering (Fig. 2, asterisk). Some MTs followed by dehydration using graded series of ethanol were very close to the nuclear membrane and propylene oxide treatments, and then embedded and far from the place of MTs origin (Fig. 2, in Epon 812. Thin sections were obtained and conarrow). There were MTs diverging from trasted with aqueous uranyl acetate and lead citrate, their apparent origin place (Fig. 2, arrowaccording to Reynolds (1%3), and examined with a Zeiss EM-10 electron microscope for nuclear analysis. heads), which were not parallel to the main bundle of MTs (Fig. 2, asterisk). A pair of RESULTS MTs is joined by their ends to another MT About 700 nuclei of E. histolytica tropho- (Fig. 2, circle). Little amounts of conzoites, in logarithmic growth phase, were densed peripheric material could be seen, studied to investigate the MTs movement and most of it was located in the upper part during the nuclear division. The stage of of this nucleus. Another section of amoebic division was determined by the shape of the trophozoites with nuclei in this stage of minuclei according to previous reports (Solis tosis, anaphase A, presented grouped MTs, et al. 1986; Orozco et al. 1988). The size forming a bundle (Fig. 3) that resembles a and shape of amoebic MTs matched the size more organized spindle (S) than the one oband shape of mammalian MTs. Figure 1 served in Fig. 2. The spindle is conformed shows a nucleus in the prophase stage of by short MTs, which seem to be joined by mitosis, which presented many radially dis- their ends. In this section no cross-secposed MTs (MT). They seemed to anchor tioned or lateral MTs were observed. The to small electron-dense bodies, with non- two types of IVs (Fig. 3, V, arrows) were electron-dense zones among them, and lo- present. There are two groups of onecated in the center of the nucleus. Some membrane IVs (Fig. 3, arrows) close to the cross-sectioned MTs were also observed nuclear envelope (NE). The already formed mitotic spindle (S) (Fig. 1, arrowheads). These electron-dense eventually grew, in anaphase B stage of mibodies might correspond to E. histolytica MTOC (M). Some of the peripheric mate- tosis, and extended along the nucleus, rial (PM) was more compact than the inter- through a large and narrow homogeneous
278
SOLIS AND BARRIOS
FIG. I. Prometaphasic plate of Enramoeba histolyrica. The MTOC (M) is electron-dense, surrounded by some peripheral chromatinic material (PM). The microtubules (MT) are arranged in a radial way. Some cross-sectioned MTs are present (arrowheads). There are two different types of intranuclear vesicles. Some of them have a thin membrane (arrow). NE, Nuclear envelope. Bar = 1 pm.
bundle of MTs (Fig. 4A). These MTs were apparently joined to themselves through their ends (Fig. 4A, inset). No crosssectioned MTs were found, probably indicating that all MTs have already reached the spindle at this stage of nuclear division. One of the spindle ends was close to the nuclear envelope, although the MTs of this spindle end did not reach the nuclear envelope. Some electron-dense material was embedded in MTs of the spindle (Fig. 4A, thin arrow). A cross-section of the mitotic spindle is shown in Fig. 4B; MTs occupy the central part of the nucleus. Most of the MTs were joined by electron-dense bridges
(Fig. 4B, inset, arrows), with no apparent regular arrangement. A group of onemembrane IVs (Fig. 4A, thick arrow) is separated from the other type of IVs. The PM was distributed uniformly in these nuclei (Figs. 4A and 4B). During the last stage of nuclear division of E. histolytica, i.e., in the telophase stage, before complete separation of the daughter nuclei, when both of them are still joined (Fig. 5, arrow), many of the MTs (MT) were still present in both nuclei and they seemed to be separated by the nuclear membrane (Fig. 5). Some nuclei had chromosome-like material (Fig. 5, arrowheads).
E. histolytica: MICROTUBIAL MOVEMENT
279
FIG. 2. Microtubule reorganization in Entamoeba histolytica nucleus. The nucleus is in anaphase A stage. The MTs (arrowheads) are diverging from a common zone (dashed arrows). A MT is close to the nuclear membrane (arrow). There is a zone with a bundle of MTs (asterisk). A pair of MTs are joined to another by their ends (circle). Bar = 1 pm.
The peripheric material (PM) is arranged in discrete small bodies. During our observations, we were not able to see the nuclei joined by a thin thread (Orozco et al. 1988); thus, we could not observe the telophasic bundle MTs. Therefore, the existence of a microtubular midbody, similar to that observed in telophasic mammalian cells (Dustin 1982), remains to be determined. DISCUSSION
In this paper, we present a more detailed image of nuclear division in E. histolytica, especially in regard to the movement of MTs during nuclear division. This proto-
zoan presents a closed, possibly intranuclear pleuromitosis (Orozco et al. 1988). The nuclear shape was taken as a reference to know approximately the stages of nuclear division of this parasite, according to previous reports (Solis et al. 1986; Orozco et al. 1988). The MTs movement at the beginning of amoebic nuclear division resembles the mitosis of the micronucleus in Didinium nasutum (Karadhazan and Raikov 1977), as inferred from the ordering of MTs in a radial way during this stage of nuclear division (Fig. 1). In metaphase, the chromosomes and the MTOC form a metaphasic plate in the same way that has been reported for Laredo strain (Gicquaud 1979),
280
SOLIS AND
BARRIOS
nucleus shows a mitotic spindle FIG. 3. Amoebic nucleus in anaphase A stage. Enfamoeba hisrolytica (S) in formation. There are grouped IVs (V). Two of them show thin surface (arrows). The MTs seem to start from the same place (M). NE, nuclear envelope. Bar = 1 pm.
although this strain is not an E. histolytica amoeba (Bhattacharya et al. 1988, among others). According to the nuclear shape, in ana-
phase A stage, the MTs might start to organize the mitotic spindle and, in this stage, some lateral and cross-sectioned MTs were present (Fig. 2, arrowheads, dashed ar-
E.
histdytica:
MICROTUBIAL
MOVEMENT
FIG. 4. (A) Entamoeba histolytica nucleus in anaphase B stage. Enlarged nucleus with joined MTs (inset) in an enlarged one-piece spindle (S). The nuclear growth might occur from rounded to pointed pole. Two groups of vesicles can be seen. One group of IV has a thin membrane (arrow). Some electron-dense material is embedded in MTs of mitotic spindle (thin arrow). Bar = 2 urn. (B) Crosssectioned MTs of the mitotic spindle. The nucleus was cross-sectioned and shows a narrow zone with MTs. Some of them are joined by electron-dense bridges (inset, arrows). Bar = 1 pm.
281
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SOLIS AND BARRIOS
FIG. 5. Sectioned mitotic spindle. The MTs (MT) of the Entamoeba hisrolytica mitotic spindle seem to be sectioned. The nuclei are still joined (arrow). One of the daughter nuclei presents chromatinic material (arrowheads). PM, Peripheric material. Bar = 2 pm.
rows). Although there were MTs very close to the membrane, we did not observe the junction of mitotic spindle MTs to the nuclear membrane in this or later stages. The
lack of MTs anchorage to the nuclear membrane has been also observed in Zridia (Cessna, 1978). After this stage, the formation (Fig. 3) and growth (Fig. 4A) of a well-
E. histolytica: MICROTUBIAL defined mitotic spindle could take place during the anaphase B stage of amoebic mitosis. In this stage, the mitotic spindle did not show MTs long enough to reach both poles, and there was not a more electrondense middle zone of interdigitating MTs. The typical mitotic spindle is composed of two halves and the middle zone presents a more electron-dense zone, due to interdigitating MTs, in most of the organisms studied (Dustin 1982; McIntosh and Koone 1989). These features might indicate that the amoebic mitotic spindle is formed by a one-piece mitotic spindle. The presence of bridges between MTs (Fig. 4B, inset, arrows) might indicate the anchoring of the MTs among themselves, as suggested by McIntosh and McDonald (1989) for typical mitotic spindles. The possible mechanism for the formation of the mitotic spindle might be the junction of the MTs by their ends (Figs. 2, circle and Fig. 4A, inset). According to the movement of MTs, the model for nuclear division of E. histolytica that we propose is as follows: (1) In prophase, the condensation of chromatinic material in the periphery of the nucleus takes place, and MTs appear, radially arranged, joined to a central body, which might be the MTOC; (2) in metaphase, the condensed chromatinic material is pulled into the center of nucleus by MTs (Solis et al., in preparation), forming a metaphasic plate; (3) in anaphase A and B, the chromosomes are carried to the nuclear poles by the growth of the mitotic spindle; the presence of electron dense material embedded in mitotic spindle (Fig. 4A, thin arrow) might suggest that the chromosome distribution takes place after the mitotic spindle is complete. (4) The telophase stage, at which the nuclei separate (karyokinesis) and the chromatinic material decondenses. This type of mitosis of E. histofytica has been suggested to be a pleuromitosis (in which the mitotic spindle is bilateral in any of the mitotic stages). Most of the protozoan related to amoeba, such as Acan-
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thamoeba (Sawyer and Griffin 1971), Hartmanella (Page, 1967), and Naegleria (Schuster 1975), undergo open or closed orthomitosis (in which the mitotic spindle is always axial). However, in E. histolytica the formation of the mitotic spindle starts when the MTs, originated from a central body, pull the chromosomes towards the central part of the nucleus. Thus, the mitotic spindle is bilateral at this moment, although with a single MTOC, which might indicate a particular pleuromitosis at the beginning of nuclear division. In regard to E. histolytica intranuclear vesicles, there are reports (Feria-Velasco and Trevino, 1972; Zaman, 1973) describing the double-membrane IV. Our results support these data, and present evidence for the existence of another type of IV. This type of IV had a regular, thin surface, with only one membrane, and it sometimes contained fibrous electron-dense material, which is morphologically similar to the nucleoplasmic material. We observed the increase of both IVs during nuclear division. However, the role of the IVs remains to be determined. ACKNOWLEDGMENTS We thank Dr. Esther Orozco for critical reviewing of this manuscript and Mr. Tomas Cruz and Mario Garcia for photographic work. This work was supported by Consejo National de Ciencia y TecnologiaMexico. REFERENCES BHATTACHARYA, S., BHATTACHARYA, A., AND DIAMOND, L. 1988. Comparison of repeated DNA from strains of Entamoeba histolytica and other Entamoeba. Molecular and Biochemical Parasitology 21, 257-262. C&ANA, D. 1978. La mitose gamogonique chez Iridia lucida (Foraminifera, Lagynidae). Annals Science Natural Zoologie s&r 12, 20, 287-320. DIAMOND, L., HARLOW, D. R., AND CUNNICK, C.
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243-251. Received 10 December 1990;accepted with revision 25 April 1991
