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First published online 14 November 2006
doi: 10.1242/jcs.03276

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Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis

Meredith S. Sagolla, Scott C. Dawson^*, Joel J. Mancuso and W. Zacheus Cande

345 LSA, Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA 94720, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. Giardia has two equivalent nuclei and a complex microtubule cytoskeleton. (A) The microtubule cytoskeleton of the trophozoite is characterized by eight flagellar axonemes and basal bodies (afl, anterior; c, caudal; pfl, posterior; vfl, ventral), the ventral adhesive disc (vd), the funis (fn) and the median body (mb) (diagramed in A). All basal bodies are between the nuclei. (B) Anti-tubulin labeling reveals the microtubule arrays, including the eight flagella, and median body. The ventral disk is more weakly stained than the other structures. Although images of Giardia are frequently presented as two-dimensional projections in dorsal perspective as in A, describing cell division in Giardia requires analysis of cell morphology in three dimensions. (C) The cell is shown in 3D from a side angle. The spatial positions of the four nuclei following nuclear division are shown arrayed along the left-right (L-R) and dorsal-ventral (D-V) axes. Three possible planes of cytokinesis are represented in C; longitudinal [anterior-posterior (A-P)], transverse (L-R) and frontal (D-V). The dorsal-posterior nuclei are marked with asterisks in C,D.

View larger version (57K): [in this window] [in a new window]   Fig. 2. During mitosis two autonomous spindles are responsible for chromosome segregation after nuclei migrate to the cell center. The centromere-specific histone cenH3 marks centromeres (cenH3:GFP in green, A-C,G-I), and centrin antibodies mark spindle poles (green, D-F,J-L). TAT1 (anti-tubulin) labels the microtubule cytoskeleton including the mitotic spindles (all cells, in red) and DAPI labels chromatin (all cells, in blue). Behavior of left (red) and right (blue) nuclei is diagramed above each stage. (A) Interphase; centromeres are discreet foci in each nucleus. (D) Interphase; centrin stains two foci in association with the flagellar basal bodies between the two nuclei. (B) Prophase; spindle microtubules appear between the two nuclei and extend around each nucleus. Centromeres are one spot on each condensed chromosome. (E) Prophase; four centrin foci are associated with the forming spindles. (C) Metaphase; following nuclear migration the nuclei become stacked along the dorsal-ventral axis. The microtubules organized into two bipolar spindles. (F) Metaphase; centrin foci are at the four spindle poles. (G) Anaphase; chromatin is segregated to spindle poles along the left-right axis of the cell with centromeres clustered at the spindle poles. (J) Anaphase; centrin remains at the spindle poles. (H) Telophase; a linear microtubule structure replaces the bipolar spindles and centromeres remain clustered. (K) Telophase; centrin foci are between the two nuclei of each daughter. (I) Cytokinesis; chromatin decondenses and the cleavage furrow forms at the anterior end, creating a heart-shaped cell. (L) Cytokinesis; centrin stains two foci between the nuclei. Cytokinesis divides the cell into left and right halves. Bar, 2 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 3. FISH to an episomal plasmid maintained in one nucleus shows nuclei are inherited with mirror image symmetry and that the nuclei never mix. (A) Interphase; the FISH probe hybridizes to the episome contained in the right nucleus. (B) In the metaphase nuclei, stacked on top of each other in the cell center, FISH signal is detected in the dorsal nucleus. (B') Projection of the first eight z-sections, dorsal nucleus, of the cell seen in B contains the FISH signal. (B") Projection of the last eight z-sections, ventral nucleus, of the cell shown in B shows the FISH signal is excluded. (C) During cytokinesis, one nucleus in each daughter is labeled with mirror image symmetry between the two cells. Red, DAPI; green, FISH. Bars, 2 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 4. As shown by TEM, the mitotic spindle is semi-open and basal bodies with axonemes are present at the four spindle poles. (A) Cross-section of a mitotic nucleus; microtubules form a sheath surrounding the nucleus. The nuclear envelope is intact except for large openings at the poles. Bar, 200 nm. (B) High-magnification view of area outlined by box in A. Cytoplasmic microtubules enter the nucleus through a large opening in the nuclear membrane. Bar, 50 nm. (B') Diagram of nuclear membrane (blue) and microtubules (red) seen in B. (C) Spindle microtubules radiate from the basal body seen in cross-section, which organizes the spindle microtubules. Bar, 300 nm. (D) Basal bodies are located between the two daughter telophase nuclei. One basal body with associated axoneme is seen in longitudinal section, the others are in cross-section. Bar, 400 nm. bb, basal body; fl, flagellum; n, nucleus.

View larger version (67K): [in this window] [in a new window]   Fig. 5. Treatment with the microtubule-stabilizing compound taxol reveals that dynamic microtubules are required for spindle integrity and proper chromosome segregation. (A) Late-anaphase cell treated with 10 µM taxol for 15 minutes. Elongated spindle microtubules are bent towards anterior of cell; lagging chromosomes remain in the spindle midzone. (B-D) Cells treated with 20 µM taxol for 1 hour. (B) Anaphase spindle morphology is normal but anaphase A chromosome segregation is delayed. (C) Anaphase spindles are broken into four half-spindle microtubule arrays. Arrows point to distal tips of broken microtubule arrays. (D) Late anaphase; spindle is bent around anterior edge of cell, with lagging chromosomes along spindle axis. Median bodies persist in mitosis and are marked with asterisks. Red, tubulin (TAT1); blue, DNA (DAPI). Bars, 2 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 6. During mitosis the flagella and basal bodies are displaced by the migrating nuclei and become associated with the spindle poles. (A) TEM of a metaphase mitotic cell shows that the caudal flagella (cfl) are displaced following nuclear migration. Bar, 1 µm. (B) Interphase; arrow marks position of basal bodies of anterior flagella. (C) Early prophase; arrow marks position of basal bodies, located at the periphery of the migrating nuclei. (D) Metaphase; the anterior flagella is wrapped around the dorsal nucleus, and the basal body is associated with the right spindle pole. Median body marked by asterisk. Bar, 2 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 7. The nuclear migration model, to the left and shaded in green, is proposed from this analysis (Figs 2, 3, 4). Each model presents three events that determine the pattern of nuclear inheritance in each division cycle: the plane of nuclear division, the plane of cytokinesis and the orientation of the daughter cells relative to one another. The schematic depicts the relative positions of the left (red) and right (blue) nuclei before and after mitosis and cytokinesis as seen from the dorsal side of the cell (see also Fig. 1). Following nuclear migration to the cell midline, the left-right (L-R) axis of nuclear division yields two daughter nuclei, one from each parental nucleus located on opposite (L-R) sides of the cell. The longitudinal plane of cytokinesis results in inheritance of nuclei with mirror image symmetry in the daughter cells. Dorsal-ventral (D-V) cytokinesis leads to inheritance of two nuclei of the same parental origin and is not supported by our data or by previous FISH studies (Ghosh et al., 2001; Yu et al., 2002). Models outlined in previous studies are presented on the right. Nuclear division was assumed to be in the D-V plane, by an unspecified mechanism, resulting in nuclei of the same parental origin residing on one side (L-R) of the cell (Filice, 1952; Ghosh et al., 2001; Yu et al., 2002; Benchimol, 2004a). Division along the longitudinal axis results in each daughter cell inheriting nuclei of the same parental origin. Cytokinesis in the frontal plane (Ghosh et al., 2001; Yu et al., 2002; Benchimol, 2004a) (Fig. 1C) results in either identical or mirror image symmetry of the two nuclei in daughter cells depending on the orientation of the cells to one another. A-P, anterior-posterior; D-D, dorsal-dorsal; D-D-V-V, dorsal-dorsal-ventral-ventral. (Yu et al., 2002; Benchimol, 2004a). (Ghosh et al., 2001; Benchimol, 2004a).