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Mitosis in primary cultures of Drosophila melanogaster larval neuroblasts

¹ Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509, USA
² Department of Biomedical Sciences, State University of New York, Albany, New York 12222, USA

View larger version (113K): [in a new window]   Fig. 1. Larval neuroblast culture. The chamber (A) is constructed from a coverslip by placing VALAP on each corner and two concentric Vaseline rings in the center. A larva is placed in the centermost ring and covered with Voltalef oil. After dissection, the brain (arrow in B) usually remains associated with the mouthparts and salivary glands, which can then be removed (C). After spreading, most cells form a monolayer (D), although some groups remain (arrows). (E) Phase-contrast image of a culture. Two neuroblasts, one in metaphase, are visible and are recognized by their large size relative to other cells. (F) Same as in E but viewed under DIC optics. See Materials and Methods for details. Bar, 20 µm (for E and F).

View larger version (116K): [in a new window]   Fig. 2. Mitosis in a neuroblast. During prophase (A) the nucleoplasm becomes granulated as the chromosomes condense. After NEB (B) the chromosomes attach to the forming spindle and quickly congress to a metaphase configuration (C). Metaphase chromosomes show little motion, and long chromosome arms are often aligned parallel to the spindle long axis (arrowheads). Sister chromatids disjoin synchronously at anaphase (D), and at this time the spindle also begins to elongate. Restitution nuclei form after the chromosomes reach the poles (F), and the spindle becomes asymmetrically positioned within the cell near the onset of telophase. Once this occurs the centrally located centrosome stops moving but spindle elongation continues by movement of the cortical centrosome (F-H). Cytokinesis is initiated during telophase (arrows in G) and progresses to form a midbody that separates the GMC from the neuroblast (arrow in H). Time is in minutes and seconds. Bar, 10 µm.

View larger version (109K): [in a new window]   Fig. 3. Mitosis in a cultured neuroblast. Similar to Fig. 2, except the spindle poles remain co-planar during prometaphase and metaphase (C). (D) Anaphase onset. (E) Late anaphase prior to spindle repositioning. (F) Telophase, just after spindle repositioning. (G,H) Cytokinesis (arrows note the furrow and midbody). Time is in minutes and seconds. Bar, 10 µm.

View larger version (53K): [in a new window]   Fig. 4. Maximum intensity projections from a 4D time-lapse recording of mitosis in a neuroblast tagged with GFP-histone. By late prophase (A) homologous chromosomes appear paired, and after NEB sister chromatids can be resolved as individual units often separated in the telomere region (B,C). Chromosomes congress rapidly (B-D), and remain in metaphase for several minutes prior to anaphase (E). During anaphase the small fourth chromosomes often lead the way poleward (inset in D-H). Time is in minutes and seconds. Bar, 5 µm; inset is 1 µm.

View larger version (37K): [in a new window]   Fig. 5. Maximum intensity projections from a 4D sequence of a dividing neuroblast containing GFP-tagged kinetochores and centrosomes. Kinetochores are labeled with GFP-fzy and centrosomes with GFP-fzr. GFP-fzy appears on kinetochores (A, arrows and small arrowheads) at NEB, while GFP-fzr is present on centrosomes (A, large arrowheads) as cells enter mitosis. After attaching to the spindle, sister kinetochores (e.g. gray and white arrows and arrowheads in A-F; filled and open circles and triangles in G) rapidly achieve a stable equatorial position. During early prometaphase some attaching kinetochores (A,B, gray arrows) exhibit a sudden rapid motion towards the proximal pole (A,B, large gray arrowhead). In the example here, the chromosome then moves away from the pole along a vector that does not intersect the distal centrosome (small white and gray arrows in B,C). These sister kinetochores then exhibited an arc-like motion that positioned them on the metaphase plate (C,D), after which they remained stationary (E,F). By comparison, sister kinetochores on chromosomes more centrally located between the centrosomes at NEB quickly become bioriented, after which they congress in one relatively smooth motion (small arrowheads in A-C; open and filled triangles in G). Time is in minutes and seconds. Bar, 10 µm. (G) Plot showing the behavior of sister kinetochores marked by the gray and white arrows/arrowheads in A-F. The positions of sister kinetochores denoted by the gray and white small arrows/arrowheads, relative to their respective poles, are plotted on the graph as filled and open circles and triangles. Letters at the top of the graph note the time points corresponding to panels (A-F).

View larger version (94K): [in a new window]   Fig. 6. Maximum intensity projections from a 4D sequence of a neuroblast expressing GFP--tubulin, as it progresses from late-prophase through cytokinesis. (A) Late prophase. (B) Early prometaphase. (C) Metaphase. (D) Anaphase. (E) Late anaphase. (F) Telophase. (G-H) Late telophase/cytokinesis. See text for details. Time is in minutes and seconds. Bar, 10 µm.

View larger version (132K): [in a new window]   Fig. 8. Secondary spindles can form in association with chromosomes attached to the centrosome-containing spindle. In this GFP--tubulin-expressing neuroblast a half-spindle is generated in association with one of the centrosomes (right-hand arrow in B). This half-spindle then grows to form a fusiform-shaped structure containing a blunt acentrosomal pole (C,D). This `secondary spindle' forms around one or more chromosomes, as shown by the fact that it contains a central band of reduced fluorescence (black arrowheads in C,D) as seen on the major spindle (white arrowheads in C,D). It then coalesces with the centrosome-containing bipolar spindle (D,E), and the cell undergoes a normal anaphase and telophase (F). Time is in minutes and seconds. Bar, 10 µm.

View larger version (87K): [in a new window]   Fig. 9. Similar to Fig. 8, except that in this example a tri-polar spindle is organized in the absence of centrosome separation. At NEB (B) the two centrosomes are closely associated and remain so throughout mitosis (arrows C-G). Regardless, two bipolar spindles ultimately form (D-F) that share a common polar region defined by the two non-separated centrosomes. Each of these spindles contains chromosomes (white arrowheads in D-F) that, during anaphase (G), segregate into three nuclei (asterisks in H). Time is in minutes and seconds. Bar, 10 µm.

View larger version (159K): [in a new window]   Fig. 7. Spindle behavior during anaphase and telophase in a neuroblast expressing GFP--tubulin. The elongating spindle remains centrally positioned until mid-anaphase (A), at which time it undergoes a sudden shift towards the cell cortex (B,C), with the future GMC cell centrosome leading (white arrowheads in A-F). After this shift, the centrally located centrosome (white arrows) stops moving (C-F) and begins to generate a more robust aster (D-F), while the spindle continues to elongate. In time-lapse recordings this elongation appears to `push' against the cortex to form a cytoplasmic protrusion into which the GMC centrosome (and its associated nucleus) move. Time is in minutes and seconds. Bar, 5 µm.