Kinetochore-dependent microtubule rescue ensures their efficient and sustained interactions in early mitosis.
Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore.Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules.Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis.
Affiliation: Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
- Saccharomyces cerevisiae/cytology*/genetics/metabolism
- Cells, Cultured
- Microtubule-Associated Proteins/metabolism
- Saccharomyces cerevisiae Proteins/metabolism
© Copyright Policy
fig7: Computer Simulation of the Initial Kinetochore-Microtubule Interaction Reveals Benefits of Kinetochore-Dependent Microtubule Rescue(A) Diagram outlines a computer simulation that recapitulates the initial KT-MT interaction. KTs locate in the vicinity of a spindle pole before centromere DNA replication (not shown in this diagram). Upon centromere replication, KTs disassemble, and centromeres move away from a pole (Kitamura et al., 2007). KTs are then reassembled and interact with MTs extended from a spindle pole. Each KT undergoing different stages of KT-MT interaction is represented by a colored circle, as explained in the text. KTs are then transported to a spindle pole by MTs and subsequently tethered at the end of short MTs in the vicinity of the pole (not shown in this diagram).(B) Example of KT-dependent MT rescue leading to MT extension (red dashed line) and interaction with other KTs further away from a spindle pole. Time zero was defined as the time of replication of the first CEN (see Supplemental Experimental Procedures). The number, next to each KT, represents the number of CEN, on which the KT has been assembled. In this example, MT rescue happened at CEN10 soon after 4.34 min, leading to interaction with CEN13 and CEN3. MT rescue also occurred distal to CEN6 soon after 8.37 min, leading to interaction with CEN8. The KT color code is shown in the text. A KT in magenta indicates MT pausing at the KT (see Figures 1Cii and 1D). For clearer presentation, one to three MTs, not associated with any CENs, were omitted at time points 4.34–4.97 min. This example of simulation is also shown in Movie S1.(C) The effect of KT-dependent MT rescue on the overall state of KT collection by MTs. One million simulations were run with each combination of the presence and absence of MT rescue at the KT and distal to the KT (Conditions 1–4). Graphs in (i, top) and (ii, top) show the distribution of median KT capture time and total KT collection time (as defined in text; time zero as defined in B), respectively. Graphs in (i, bottom) and (ii, bottom) show relative difference between the presence and absence of KT-dependent MT rescue (rescue both at KT and distal to the KT), as defined by the indicated formulas. Bins along the x axis were 0.02 min (i, top), 0.04 min (i, bottom), 0.08 min (ii, top), and 0.64 min (ii, bottom).(D) MT rescue distal to the KT is particularly useful to collect other KTs that have drifted further away from a spindle pole. One million simulations were run in each condition. Graph (top) shows the distribution of the positions (distance from a spindle pole) for KT captures by rescued MTs (along the extended MT region following rescue), in Condition 3 (rescue only at the KT) and Condition 4 (rescue both at the KT and distal to the KT). The binning along the x axis was 0.03 μm. The heat map (bottom) shows the spatial distribution in the nucleus of relative difference in the number of KT captures by rescued MTs (as defined above) between Conditions 3 and 4 as defined by the formula. The circle (gray) represents the nuclear envelope. KT capture events along the y axis were evaluated to project the relative difference to the x–z plane.See also Figure S6.
KTs localize in the vicinity of a spindle pole in the G1 phase, but upon centromere DNA replication they disassemble and centromeres move away from the pole (Kitamura et al., 2007). In our simulation, we assumed that this happened to 16 centromeres of S. cerevisiae over 7.5 min, following their defined replication timing (Yabuki et al., 2002). One minute after replication of each centromere, the KT was reassembled (Figure 7A, red dot) and subsequently caught on the lateral surface of a MT extending from a spindle pole (green dot) (Kitamura et al., 2007); this capture process was facilitated by short MTs transiently generated at the KT (dashed line from a yellow dot) (Kitamura et al., 2010). KTs were then transported toward a spindle pole by sliding along a MT (Figure 7A, green dot) and, subsequently, by end-on pulling (blue dot) once end-on attachment was established (Tanaka et al., 2007). Most parameter values for MT dynamics and centromere motions were defined in our previous studies of live-cell imaging and electron tomography (Kitamura et al., 2007, 2010; Tanaka et al., 2005, 2007).