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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01213

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Drosophila Klp67A is required for proper chromosome congression and segregation during meiosis I

Matthew S. Savoian^1,*, Melanie K. Gatt^1,*, Maria G. Riparbelli², Giuliano Callaini² and David M. Glover^1,

¹ Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK
² Department of Evolutionary Biology, University of Siena, Siena, Via Aldo Moro, 2-53100, Italy

View larger version (43K): [in a new window]   Fig. 1. Testes isolated from P-element-mediated mutants have diminished Klp67A levels. Western blot showing the different allelic combinations used in this study. A band of the expected molecular weight of 92 kDa was present in Oregon R testes but was greatly diminished in homozygous mutant third instar larvae and not detectable in homozygous pharate adults or hemizygous or heteroallelic individuals at any stage. -tubulin is shown as a loading control.

View larger version (84K): [in a new window]   Fig. 2. klp67A mutants exhibit karyokinetic and cytokinetic failure during meiosis. (A) Phase-contrast image of onion-stage spermatids taken from a klp67A mutant. Unlike wild-type control spermatids which contain a single nucleus and phase dense Nebenkern, those in the mutants may contain two nuclei of equal or varying sizes, indicative of cytokinetic failure and in the latter case, of segregation defects as well. (B-I) A gallery of spindles taken from the wild type and klp67A mutant primary spermatocytes; tubulin is shown in green, DNA in blue and the centriole marker, Centrin in red. In klp67A mutants the centrosomes separate and form highly aberrant bipolar spindles. Unlike control spindles seen during metaphase (B) and anaphase (E), those in klp67A mutants are poorly organised (C,D,F-I). Ectopic MTs are present in the regions of the spindle as well as in the surrounding cytoplasm. The astral MTs are more robust than in wild-type cells and may form bundles that run around the cell periphery (C). The chromosomes sometimes appear entangled in the increased numbers of MTs found in the spindle. Kinetochore fibres in klp67A mutants could be hard to discern and often have an elongated and bent morphology (arrowheads in C). In contrast to wild-type anaphase/telophase cells (E) mutants lack a distinct central spindle but are filled with ectopic MTs and abnormal MT structures (F-I). Note how the chromosomes appear to segregate in unequal masses and initiate decondensation at non-polar locations. Some chromosomes may be linked together by non-centrosomal MT bundles (arrow in H) or have MT bundles oriented in opposite directions, suggestive of merotelic malorientation (arrowhead in I). Bars, 10 µm.

View larger version (132K): [in a new window]   Fig. 3. Chromosomes in klp67A mutant primary spermatocytes are abnormally dynamic and undergo exaggerated periods of reorientation during prometaphase. (A) Time-line comparing karyokinetic events in wild-type Oregon R primary spermatocytes and in the two independent P-element-mediated klp67A mutant strains examined. The time is in minutes relative to prometaphase onset. Red bars indicate the reorientation period and anaphase onset is indicated by the blunted ends of each line. Although the mutants enter anaphase with similar timing to the controls, their chromosomes reorient for substantially longer. (B) Selected images from a time-lapse sequence during prometaphase in a klp67A mutant. The black line (00 timepoint) indicates the long axis of the cell. The two homologous dyads comprising the bivalent of interest are pseudo-coloured red and blue. This chromosome continues to reorient for over 22 minutes before moving out of the plane of focus. During this time it makes a few poleward ingressions but generally remains near the spindle equator. Time in upper right of each panel is in seconds relative to an arbitrary start time. Bar, 10 µm.

View larger version (111K): [in a new window]   Fig. 4. Dyads in klp67A mutants segregate at greatly reduced velocities. (A) Selected images from a time-lapse sequence showing a wild-type cell during anaphase. The homologous dyads have been pseudo-coloured and their anaphase movements plotted (B). The average maximal velocity for each of the dyads of interest is indicated. Once wild-type dyads initiate poleward motion it continues uninterrupted until they reach their poles. (A') Selected frames from a time-lapse sequence during anaphase in a klp67A mutant cell. The chromosomes have been coloured as above. (A') Prior to anaphase onset the dyads appear `twisted' relative to one another (–128 second time point). Following disjunction, the dyads in this cell separate and initiate movement to their respective poles. One dyad (blue) travels out of the plane of focus at 200 seconds. The red dyad begins its poleward migration and then abruptly halts at 152 seconds and remains stationary relative to the spindle pole, and exhibits no further poleward movement before decondensing. (B') Kinetic plot and average maximal velocities for the dyads shown in (A'). Time in seconds relative to anaphase onset is given in the right-hand corner. Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 5. klp67A mutants form unstable metaphase plates and have merotelically maloriented chromosomes. (A-F) Selected frames from a time-lapse sequence showing metaphase (A) and anaphase (B-F) in a klp67A mutant primary spermatocyte. Prior to anaphase onset, chromosome IV (black arrowhead) travels away from the metaphase plate before moving out of the plane of focus near the lower pole. The adjacent bivalents are abnormally resolved and their individual chromatids are visible. As anaphase ensues, one bivalent (pseudo-coloured) disjoins. Both (red) chromatids of one dyad remain associated and travel to the lower, proximal pole as predicted. The homologous (blue) dyad separates into its two component chromatids, with one chromatid moving towards the expected upper pole and out of focus (D-F), whereas its sister chromatid (white arrowhead) begins to move towards the upper pole (D) then halts before rapidly changing direction and travelling to the lower, incorrect pole (E,F), resulting in aneuploidy. Time is in seconds relative to anaphase onset. Bar in C, 10 µm; F, 2 µm.

View larger version (122K): [in a new window]   Fig. 6. Klp67A associates with pre-anaphase kinetochores and the central spindle during meiosis I. (A,B and D-F) Microtubules are shown in green, Klp67A in red and DNA in blue. During interphase and prophase (A) Klp67A localises exclusively to the nucleus where it remains until prometaphase. At this time, the protein redistributes to the cytoplasm, but is excluded from regions occupied by spindle MTs (B,C). Klp67A is also detected as pairs of opposing spots on the chromosomes (B,C, arrowheads), that interact with presumptive kinetochore fibres (D). Co-localisation studies (G) using anti-CID antibodies to label centromeres or GFP-Polo-tagged outer kinetochore plates, suggest that Klp67A is a new component of pre-anaphase kinetochores (H). Following anaphase onset Klp67A redistributes to an equatorial band on the central spindle (E), where it remains during cytokinesis. During this time Klp67A is still detected in the cytoplasmic pool, and as telophase initiates, in the reforming nuclei (F). Bar in D, 2 µm; F, 10 µm; G, 1 µm.