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Kinetochore-dependent microtubule rescue ensures their efficient and sustained interactions in early mitosis.

Gandhi SR, Gierliński M, Mino A, Tanaka K, Kitamura E, Clayton L, Tanaka TU - Dev. Cell (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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Stu2 Is Closely Associated with a Fraction of Kip3 during Its Transport from the Kinetochore along a Microtubule(A) PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP STU2-4×mCherry KIP3-3×GFP (T6038) cells were treated as in Figure 1C. Images were collected every 10 s using CFP, GFP, and mCherry channels. (i) Time-lapse images showing a representative example of colocalization of Stu2 and Kip3 during transport from the KT to the MT plus end. Scale bar, 1 μm. (ii) Graph showing 11 events of Stu2 transports during which Stu2 and Kip3 did (blue) or did not (orange) show colocalization.(B) (i) Schematic diagram showing the principle of the BiFC assay. (ii) Representative time-lapse images of a BiFC signal, generated by the close association between Stu2-VN and Kip3-VC, which moved along the MT toward its plus end. PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP CFP-TUB1 STU2-VN KIP3-VC cells (T6736) were treated as in Figure 1C. Images were collected every 7 s using CFP and YFP (for BiFC) channels. Scale bar, 1 μm.(C) The N-terminal half of Kip3 associates with the C-terminal half of Stu2 in a two-hybrid assay. The constructs, shown as diagrams (top), were fused with a DNA-binding domain (BD) or an activation domain (AD); amino acid residue numbers are in red. Equal numbers of cells, expressing the indicated combinations of constructs, were spotted on histidine drop-out plates and incubated for 48 hr (bottom).(D) Stu2 binds to Kip3 in vitro. MBP-Kip3 or MBP alone was immobilized on amylose beads. Coomassie blue lanes show the input proteins. After incubation with yeast extract from STU2-HA and BIK1-HA cells, HA-tagged proteins bound to the beads were detected by western blot. Input lanes were 1/250 of reaction.See also Figure S4.
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fig5: Stu2 Is Closely Associated with a Fraction of Kip3 during Its Transport from the Kinetochore along a Microtubule(A) PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP STU2-4×mCherry KIP3-3×GFP (T6038) cells were treated as in Figure 1C. Images were collected every 10 s using CFP, GFP, and mCherry channels. (i) Time-lapse images showing a representative example of colocalization of Stu2 and Kip3 during transport from the KT to the MT plus end. Scale bar, 1 μm. (ii) Graph showing 11 events of Stu2 transports during which Stu2 and Kip3 did (blue) or did not (orange) show colocalization.(B) (i) Schematic diagram showing the principle of the BiFC assay. (ii) Representative time-lapse images of a BiFC signal, generated by the close association between Stu2-VN and Kip3-VC, which moved along the MT toward its plus end. PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP CFP-TUB1 STU2-VN KIP3-VC cells (T6736) were treated as in Figure 1C. Images were collected every 7 s using CFP and YFP (for BiFC) channels. Scale bar, 1 μm.(C) The N-terminal half of Kip3 associates with the C-terminal half of Stu2 in a two-hybrid assay. The constructs, shown as diagrams (top), were fused with a DNA-binding domain (BD) or an activation domain (AD); amino acid residue numbers are in red. Equal numbers of cells, expressing the indicated combinations of constructs, were spotted on histidine drop-out plates and incubated for 48 hr (bottom).(D) Stu2 binds to Kip3 in vitro. MBP-Kip3 or MBP alone was immobilized on amylose beads. Coomassie blue lanes show the input proteins. After incubation with yeast extract from STU2-HA and BIK1-HA cells, HA-tagged proteins bound to the beads were detected by western blot. Input lanes were 1/250 of reaction.See also Figure S4.

Mentions: Could Stu2 be a cargo that is transported by Kip3 along a MT? If so, we expect that Stu2 and Kip3 would move together from CEN3 along a MT. Colocalization of Stu2 and Kip3 signals was transiently observed at CEN3 (Figure 5Ai, 80 s). While Stu2 was transported from CEN3 along a MT toward its plus end (90–170 s), Stu2 signals continued to colocalize with a fraction of Kip3 signals. During 11 events of Stu2 transport from CEN3 toward the MT plus end, Stu2 signals colocalized continuously with Kip3 signals at most of the time points (20–130 s duration) (Figure 5Aii). In all 11 events of Stu2 transport, the relevant MTs showed rescue upon arrival of Stu2 at the MT plus end (e.g., Figure 5Ai, 170–180 s). While most Stu2 signals colocalized with a fraction of Kip3 signals during transport (Figure 5Ai, magenta arrowheads), other Kip3 signals showed movement along a MT without colocalization with Stu2 (white arrowheads).


Kinetochore-dependent microtubule rescue ensures their efficient and sustained interactions in early mitosis.

Gandhi SR, Gierliński M, Mino A, Tanaka K, Kitamura E, Clayton L, Tanaka TU - Dev. Cell (2011)

Stu2 Is Closely Associated with a Fraction of Kip3 during Its Transport from the Kinetochore along a Microtubule(A) PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP STU2-4×mCherry KIP3-3×GFP (T6038) cells were treated as in Figure 1C. Images were collected every 10 s using CFP, GFP, and mCherry channels. (i) Time-lapse images showing a representative example of colocalization of Stu2 and Kip3 during transport from the KT to the MT plus end. Scale bar, 1 μm. (ii) Graph showing 11 events of Stu2 transports during which Stu2 and Kip3 did (blue) or did not (orange) show colocalization.(B) (i) Schematic diagram showing the principle of the BiFC assay. (ii) Representative time-lapse images of a BiFC signal, generated by the close association between Stu2-VN and Kip3-VC, which moved along the MT toward its plus end. PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP CFP-TUB1 STU2-VN KIP3-VC cells (T6736) were treated as in Figure 1C. Images were collected every 7 s using CFP and YFP (for BiFC) channels. Scale bar, 1 μm.(C) The N-terminal half of Kip3 associates with the C-terminal half of Stu2 in a two-hybrid assay. The constructs, shown as diagrams (top), were fused with a DNA-binding domain (BD) or an activation domain (AD); amino acid residue numbers are in red. Equal numbers of cells, expressing the indicated combinations of constructs, were spotted on histidine drop-out plates and incubated for 48 hr (bottom).(D) Stu2 binds to Kip3 in vitro. MBP-Kip3 or MBP alone was immobilized on amylose beads. Coomassie blue lanes show the input proteins. After incubation with yeast extract from STU2-HA and BIK1-HA cells, HA-tagged proteins bound to the beads were detected by western blot. Input lanes were 1/250 of reaction.See also Figure S4.
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fig5: Stu2 Is Closely Associated with a Fraction of Kip3 during Its Transport from the Kinetochore along a Microtubule(A) PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP STU2-4×mCherry KIP3-3×GFP (T6038) cells were treated as in Figure 1C. Images were collected every 10 s using CFP, GFP, and mCherry channels. (i) Time-lapse images showing a representative example of colocalization of Stu2 and Kip3 during transport from the KT to the MT plus end. Scale bar, 1 μm. (ii) Graph showing 11 events of Stu2 transports during which Stu2 and Kip3 did (blue) or did not (orange) show colocalization.(B) (i) Schematic diagram showing the principle of the BiFC assay. (ii) Representative time-lapse images of a BiFC signal, generated by the close association between Stu2-VN and Kip3-VC, which moved along the MT toward its plus end. PMET3-CDC20 PGAL-CEN3-tetOs TetR-3×CFP CFP-TUB1 STU2-VN KIP3-VC cells (T6736) were treated as in Figure 1C. Images were collected every 7 s using CFP and YFP (for BiFC) channels. Scale bar, 1 μm.(C) The N-terminal half of Kip3 associates with the C-terminal half of Stu2 in a two-hybrid assay. The constructs, shown as diagrams (top), were fused with a DNA-binding domain (BD) or an activation domain (AD); amino acid residue numbers are in red. Equal numbers of cells, expressing the indicated combinations of constructs, were spotted on histidine drop-out plates and incubated for 48 hr (bottom).(D) Stu2 binds to Kip3 in vitro. MBP-Kip3 or MBP alone was immobilized on amylose beads. Coomassie blue lanes show the input proteins. After incubation with yeast extract from STU2-HA and BIK1-HA cells, HA-tagged proteins bound to the beads were detected by western blot. Input lanes were 1/250 of reaction.See also Figure S4.
Mentions: Could Stu2 be a cargo that is transported by Kip3 along a MT? If so, we expect that Stu2 and Kip3 would move together from CEN3 along a MT. Colocalization of Stu2 and Kip3 signals was transiently observed at CEN3 (Figure 5Ai, 80 s). While Stu2 was transported from CEN3 along a MT toward its plus end (90–170 s), Stu2 signals continued to colocalize with a fraction of Kip3 signals. During 11 events of Stu2 transport from CEN3 toward the MT plus end, Stu2 signals colocalized continuously with Kip3 signals at most of the time points (20–130 s duration) (Figure 5Aii). In all 11 events of Stu2 transport, the relevant MTs showed rescue upon arrival of Stu2 at the MT plus end (e.g., Figure 5Ai, 170–180 s). While most Stu2 signals colocalized with a fraction of Kip3 signals during transport (Figure 5Ai, magenta arrowheads), other Kip3 signals showed movement along a MT without colocalization with Stu2 (white arrowheads).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Show MeSH
Related in: MedlinePlus