Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

The ultrastructure of mitosis inIsochrysis galbana parke (Prymnesiophyceae)

Summary Mitosis and cytokinesis have been studied in the flagellate algaIsochrysis galbana Parke (Prymnesiophyceae). Nuclear division is preceded by replication of the flagella and haptonema, the Golgi body and the chloroplast; fission in the chloroplast occurs in the region of the pyrenoid. During prophase, spindle microtubules radiating from two ill-defined poles are formed. The nuclear envelope breaks down and the chromatin condenses. At metaphase the spindle is fully developed, some pole-to-pole microtubules passing through the well-defined chromatin plate, others terminating at it. No kinetochores or individual chromosomes were observed. By late metaphase, many Golgi-derived vesicles may be seen against the two poleward faces of the metaphase plate. During anaphase, the two daughter masses of chromatin move towards the poles. In early telophase, the nuclear envelope of each daughter nucleus is complete only on the side towards the adjacent chloroplast, remaining open on the interzonal side. However, during telophase each nucleus becomes reorientated so that it lies lateral to the long axis of the spindle and with its open side towards the chloroplasts. By late telophase, each new nuclear envelope is complete and confluence with the adjacent chloroplast ER established.Cytokinesis and subsequent segregation of the daughter cells are effected by the dilation of Golgi- and ER-derived vesicles in the interzonal region. No microtubular structures are involved. Comparisons with the results from other studies of mitosis in members of thePrymnesiophyceae show that they all have a number of features in common, but that there are differences in detail between species.     

Particularities of mitochondrial structure in parasitic protists (Apicomplexa and Kinetoplastida)

Without mitochondria, eukaryotic cells would depend entirely on anaerobic glycolysis for ATP generation. This also holds true for protists, both free-living and parasitic. Parasitic protists include agents of human and animal diseases that have a huge impact on world populations. In the phylum Apicomplexa, several species of Plasmodium cause malaria, whereas Toxoplasma gondii is a cosmopolite parasite found on all continents. Flagellates of the order Kinetoplastida include the genera Leishmania and Trypanosoma causative agents of human leishmaniasis and (depending on the species) African trypanosomiasis and Chagas disease. Although clearly distinct in many aspects, the members of these two groups bear a single and usually well developed mitochondrion. The single mitochondrion of Apicomplexa has a dense matrix and many cristae with a circular profile. The organelle is even more peculiar in the order Kinetoplastida, exhibiting a condensed network of DNA at a specific position, always close to the flagellar basal body. This arrangement is known as Kinetoplast and the name of the order derived from it. Kinetoplastids also bear glycosomes, peroxisomes that concentrate enzymes of the glycolytic cycle. Mitochondrial volume and activity is maximum when glycosomal is low and vice versa. In both Apicomplexa and trypanosomatids, mitochondria show particularities that are absent in other eukaryotic organisms. These peculiar features make them an attractive target for therapeutic drugs for the diseases they cause.     

The ultrastructure of mitosis during sporangiogenesis inWoronina pythii (Plasmodiophoromycetes)

Summary Mitotic divisions during sporangiogenous plasmodial cleavage inWoronina pythii were studied with transmission electron microscopy. We conclude that these nuclear divisions (e.g., transitional nuclear division, and sporangial mitoses) share basic similarities with the cruciform nuclear divisions inW. pythii and other plasmo-diophoraceous taxa. The major distinction appeared to be the absence of nucleoli during sporangial mitosis and the presence of nucleoli during cruciform nuclear division. The similarities were especially evident with regard to nuclear envelope breakdown and reformation. The mitotic divisions during formation of sporangia were centric, and closed with polar fenestrae, and characterized by the formation of intranuclear membranous vesicles. During metaphase, anaphase, and telophase, these vesicles appeard to bleb from the inner membrane of the original nuclear envelope and appeared to coalesce on the surface of the separating chromatin masses. By late telophase, the formation of new daughter nuclear envelopes was complete, and original nuclear envelope was fragmented. New observation pertinent to the mechanisms of mitosis in thePlasmodiophoromycetes include a evidence for the incorporation of membrane fragments of the original nuclear envelope into new daughter nuclear envelopes, and b the change in orientation of paired centrioles during sporangial mitosis.     

Nucleoporins: leaving the nuclear pore complex for a successful mitosis

The nuclear envelope (NE) separates the cytoplasm and the cell nucleus of interphase eukaryotic cells and nuclear pore complexes (NPCs) mediate the macromolecular exchange between these two compartments. The NE and the NPCs of vertebrate cells disassemble during prophase and the nuclear pore proteins (nucleoporins) are distributed within the mitotic cytoplasm. For an increasing number of them active mitotic functions have been assigned over the past few years. Nucleoporins are participating in spindle assembly, kinetochore organisation, and the spindle assembly checkpoint, all processes that control chromosome segregation and are important for maintenance of genome integrity. But nucleoporins are also engaged in early and late mitotic events, such as centrosome positioning and cytokinesis. Here we will highlight recent progress in deciphering the roles for nucleoporins in the distinct steps of mitosis.     

Preprophasic establishment of division polarity in monoplastidic mitosis of hornworts

Summary Preprophase in the monoplastidic mitotic cells ofPhaeoceros andNotothylas is characterized by the establishment of a division site in the absence of a typical preprophase band. The future cytokinetic plane is predicted by plastid orientation and development of an elaborate preprophasic microtubule system perpendicular to the division plane. Division of the single plastid is initiated early in preprophase and the constricting plastid migrates to a position perpendicular to the future plane of division. Plastid orientation assures that division of the plastid by mid-constriction will result in distribution of a plastid to each daughter cell. Microtubules parallel the long axis of the plastid and are most numerous adjacent to the nucleus which becomes elongated in the future spindle axis. We conclude that the division site is a fundamental component of the cytokinetic apparatus involved in the determination of cleavage plane prior to nuclear division.     

The kinetochore fiber structure in the acentric spindles of the green algaOedogonium

Summary The microtubule (MT) arrangement in three kinetochore fibers in the acentric spindles of the green algaOedogonium cardiacum were reconstructed from serial sections of prometaphase and metaphase cells. The majority of the MTs attached to the kinetochore (kMTs) are relatively short, extending less than a third of the distance to the putative spindle pole region, and none extended the full distance. Fine filaments and a matrix described earlier (Schibler andPickett-Heaps 1980) were associated with the MTs all along the fibers. Live cells ofOedogonium were also studied by time lapse cinematography for correlation with the ultrastructural observations. Late prometaphase and metaphase kinetochore fibers appear to move independently as if unattached at their poleward ends. These observations suggest that kinetochore fibers inOedogonium are not attached to a specific pole structure from late prometaphase until the inception of anaphase. The results are discussed with reference to spindle structure and function in general.     

Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819-834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-N-oxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150(Glued) depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phospho-epitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.     

Leishmania (Kinetoplastida): species typing with isoenzyme and PCR-RFLP from cutaneous leishmaniasis patients in Ethiopia

Cutaneous leishmaniasis (CL) is an increasing public health problem in Ethiopia. There is a concern that it is spreading with increased incidence. In this study, we used isoenzyme electrophoresis and internal transcribed spacer one (ITS1) PCR-RFLP techniques to identify Leishmania species from CL patients in Ethiopia. We obtained isolates from 55 localized cutaneous leishmaniasis (LCL), 3 diffused cutaneous leishmaniasis (DCL) and 36 biopsy samples from 34 LCL and 2 DCL cases from All Africa Leprosy and Tuberculosis Rehabilitation and Training Center (ALERT) and clinically diagnosed CL cases from Ochollo village. Both isoenzyme and ITS1 PCR-RFLP techniques showed that Leishmania aethiopica (L. aethiopica) was the aetiologic agent in all cases. Our study also showed that ITS1 PCR-RFLP could identify Leishmania species from biopsy samples and suggests the method could be used for epidemiological surveillance of leishmaniasis in Ethiopia and for species-specific diagnosis.     

In situ visualization of a simple bipartite kinetochore with a single microtubule attachment in Giardia intestinalis (Metamonada)

To understand general features in evolution of kinetochore organization, investigating a wide range of mitotic mechanisms in various non-model eukaryotes is necessary. A binucleate flagellate Giardia intestinalis is a representative of highly divergent eukaryotic lineage of Metamonads. FIB/SEM tomography was used to investigate ultrastructural details of its mitotic architecture, including kinetochores. Giardia undergoes semi-open mitosis, with the nuclear envelope remaining intact except for polar fenestrae, allowing microtubules to enter the nucleoplasm. At the onset of mitosis, the nuclear envelope bends inward, forming a concave depression at the spindle poles. Spindle microtubules emanate from a cytoplasmic fuzzy microtubule organizing center near the flagellar basal bodies. Kinetochoral microtubules enter the nucleoplasm and bind to kinetochores. A small bipartite kinetochore composed of a dense inner disk, approximately 46 nm in diameter, and a two-armed outer fork, is attached to just one microtubule. To our knowledge, this is the first in situ evidence of a one-microtubule attachment to a kinetochore, which could represent a basic eukaryotic situation.     

Monoplastidic mitosis in the mossTimmiella barbuloides (Bryophyta)

Summary Mitotic cell division of monoplastidic sporogones was investigated in the mossTimmiella barbuloides (Brid.) Moenk. (Pottiales, Bryophyta) by TEM. Division polarity of sporogones is established by the interphase position of the single oblong cup-shaped plastid, which is orientated with its long axis parallel to one of the cell walls. In preprophase the plastid elongates and its extremities bend at right angles. Plastid growth is directed by microtubules and accompanied by plastid tubules. The plastid begins the process of duplication by constricting centrally in the plane of the future cytokinetic septum. There is no preprophase band of microtubules at the division site. The large central nucleus becomes fusiform and aligned parallel to the main plastid axis. By the end of prophase the daughter plastids are positioned at the opposite poles of the nucleus where they probably function as nucleating or organizing centres for the spindle microtubules. Metaphase and anaphase spindles contain long sheets of ER. Cytokinesis involves the formation of a well developed phragmoplast.Abbreviations TEM transmission electron microscopy - PPB preprophase band of microtubules - ER endoplasmic reticulum     

Ultrastructure of mitosis in the aphid-pathogenic fungusErynia neoaphidis

Summary An account of mitosis in the aphid-pathogenic, entomophthoraceous fungusErynia neoaphidis is presented. The mitotic apparatus is characterized by a closed, intranuclear, polarized spindle. Chromosomes are permanently attached by kinetochore microtubules (kcMTs) to the poles during mitosis. The spindle develops as the spindle pole bodies migrate and separate. At metaphase the eccentric spindle contains only kcMTs and is located in a relatively chromatinfree zone. Paired sister kinetochores are arranged in a broad metaphase plate. During anaphase kcMTs shorten, astral and nonchromosomal microtubules develop and elongate and the interpolar distance increases.     

Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida

Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP^2–3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Proliferation and differentiation of intestinal epithelial cells during development of Barbus conchonius (Teleostei,Cyprinidae)

Summary The processes of proliferation, cell division and differentiation of intestinal epithelial cells have been studied during development of the fish, Barbus conchonius. On the 3rd day, nearly all cells of the presumptive gut proliferate. Once the intestinal epithelium begins to differentiate, a decreasing percentage of proliferative cells can be found. On the 7th day, when intestinal folds start to develop, the proliferative cells become restricted to the future basal parts of the folds.Ultrastructural examination of ³H-thymidine-labeled cells and mitotic cells of 6-day-old larvae shows that functional enterocytes are proliferative. The same feature is suggested for older fish. Proliferating undifferentiated dark cells, characterized by many free ribosomes and a few organelles, are also present in the intestinal epithelium of larval fish; they are considered to be stem cells, mainly for goblet cells. Proliferating goblet cells and enteroendocrine cells were not observed. The latter cell type is scarce and has a long turnover time.A common feature of all these dividing cells is the presence of isolated spherical to cylindrical lamellar structures which may have lost contact with the cell membrane during prophase; they probably regain this contact by fusion with the cell membrane at the end of mitosis.     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

Fine Structure of Bodo curvifilus Griessmann (Kinetoplastida: Bodonidae)*

SYNOPSIS. Bodo curvifilus Griessmann conforms in its fine structure to the criteria proposed for the genus Bodo, including the presence of subpellicular microtubules, a single large kinetoplast-mitochondrion, emergence of the 2 heterodynamic flagella from a subapical flagellar pocket, and the presence of a paraxial rod associated with the axoneme of each flagellum. B. curvifilus possesses cytoplasmic bodies which resemble endosymbiotic bacteria. These are similar to those found in Bodo saltans. Bodo curvifilus can be distinguished ultrastructurally from Bodo caudatus and B. saltans by the presence in B. curvifilus of a hitherto unreported structure, “the microtubular prism,”consisting of a bundle of 19 microtubules. In cross section, 15 of these microtubules form a cross-linked prismatic array. This microtubular bundle originates near the flagellar pocket and extends for several micrometers into the body of the organism where it follows the periphery of the cell and the long finger-like projections of the kinetoplast-mitochondrion.     

Interplay between the DNA Damage Proteins MDC1 and ATM in the Regulation of the Spindle Assembly Checkpoint

To avoid genomic instability, cells have developed surveillance mechanisms such as the spindle assembly checkpoint (SAC) and the DNA damage response. ATM and MDC1 are central players of the cellular response to DNA double-strand breaks. Here, we identify a new role for these proteins in the regulation of mitotic progression and in SAC activation. MDC1 localizes at mitotic kinetochores following SAC activation in an ATM-dependent manner. ATM phosphorylates histone H2AX at mitotic kinetochores, and this phosphorylation is required for MDC1 localization at kinetochores. ATM and MDC1 are needed for kinetochore localization of the inhibitory mitotic checkpoint complex components, Mad2 and Cdc20, and for the maintenance of the mitotic checkpoint complex integrity. This probably relies on the interaction of MDC1 with the MCC. In this work, we have established that ATM and MDC1 maintain genomic stability not only by controlling the DNA damage response, but also by regulating SAC activation, providing an important link between these two essential biological processes.