Fig. 3. Confocal serial section reconstructions of cells in early prometaphase I, showing kinetochore-microtubule associations of autosomes. (A) Prometaphase cell labelled with antibodies against tubulin. Bar, 10 µm. (B) Higher magnification image of cell in (A). The closed arrowhead points to a bivalent with associations to only one pole. Open arrowheads point to a bipolar connection made by a single half-bivalent. The sister half-bivalent has microtubule associations only to the top pole. The arrow points to a half-bivalent with bidirectional microtubule associations, only one of which points towards a pole. Bar, 5 µm. (C-E) Triple-labelled prometaphase cells, stained for acetylated tubulin (green), tyrosinated tubulin (red) and chromosomes (green). Co-localization of acetylated and tyrosinated tubulin would result in a yellow or orange image. (C) Arrowheads point to microtubule associations with a bivalent that has connections to both poles. The arrow points to a kinetochore fibre pointed away from the poles. Bar, 5 µm. (D) Different series of sections from the same cell shown in (C). The closed arrowhead points to a bivalent with attachments only to one pole: notice the two fibres coming from one half-bivalent, one from each sister chromatid. Bar, 5 µm. (E) Arrowheads point to bivalents that have reached the central spindle and established bipolar connections. Bar, 5 µm. (F,G) Stereo images of a prometaphase cell showing the arrangements of microtubules and chromosomes. Bar, 10 µm.

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