Fig. 1.

A single MT accommodates two KTs with lateral attachment but only one KT with sustained end-on attachment. (A) Diagrams explaining how a KT is captured and transported by a MT in eukaryotic cells. The KT initially interacts with the lateral surface of a single MT (lateral attachment), which extends from a spindle pole; the KT is then transported along the MT lateral surface towards a spindle pole by sliding (lateral sliding). Subsequently, the KT is tethered at the end of a single MT (end-on attachment) and transported polewards as the MT shrinks (end-on pulling) (Tanaka, 2010). (B) Diagrams summarizing the interaction between a single MT and two KTs [two pairs of sister KTs on the indicated centromeres (CENs)]. Two indicated CENs were under control of the GAL promoter and visualized as fluorescent dots; these were inactivated, and subsequently reactivated, as in Fig. S1A, to study their interaction with a MT in detail. After both CENs were loaded on the lateral surface of a single MT, they showed sliding along the MT. In some cases, one CEN underwent conversion into end-on attachment, was transported by end-on pulling, and subsequently came into contact with the other CEN. Then, after brief co-transport, the CEN originally proximal to the spindle pole showed detachment. Note that either or both CENs could reach a spindle pole from any of these stages without going through subsequent stages. (C) Representative example in which two KTs showed lateral sliding along a single MT. Cells (T6519) carry P_GAL-CEN3-tetOs (replacing CEN15 on chromosome XV) TetR-3×CFP P_GAL-CEN3-lacO (replacing CEN3 on chromosome III) GFP-LacI YFP-TUB1 P_MET3-CDC20, where tetOs are tetracycline operators, TetR is the tetracycline repressor, lacOs are lactose operators, and LacI is the lactose repressor. The GFP and YFP signals were collected together (green) while CFP signals were acquired separately (red). These cells were treated as in Fig. S1A, i.e. were cultured overnight in methionine drop-out medium with raffinose, treated with a mating hormone for 2.5 h (to arrest cells in G1 phase), and released into fresh media with raffinose, galactose and 2 mM methionine (for Cdc20 depletion and P_GAL-CEN inactivation). After 4 h, cells were suspended in synthetic complete medium containing glucose and methionine (to reactivate P_GAL-CEN). After 10 min incubation, images were acquired every 20 s for 30 min. Time zero is set arbitrarily. Ch III, chromosome III; Ch XV, chromosome XV. The left panel shows a representative cell while the right shows the profile of KT movement, i.e. graphs of length of the MT that interacted with the two labeled CENs, and positions of those two CENs (distance from a spindle pole; dashed red and green lines represent CENs not on the MT, while solid red and green lines represent CENs on the MT). See Movie 1. T9717 cells (see D) showed similar results (Fig. S1B). (D) Representative example where a laterally attached KT showed detachment after coming into contact with an end-on attached KT. Cells (T9717) with the same genotype as T6519 (see C), except for carrying GFP-TUB1 instead of YFP-TUB1, were treated as in C, and images (GFP and CFP signals) were acquired every 13 s. The graph on right shows the MT length and CEN positions as in C. See Movie 2. Another example of KT detachment is shown in Fig. S1B.