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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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A Shrinking Microtubule, Whose Lateral Surface Is Associated with a Kinetochore, Shows Rescue before or after Its Plus End Reaches the Kinetochore(A) Experimental system to analyze the initial interaction between a KT (on CEN3) and an individual spindle-pole MT (Tanaka et al., 2005).(B) Schematic diagram showing possible fates of a shrinking MT: when a MT plus end catches up with CEN3 on its lateral side, either end-on attachment/pulling of CEN3 occurs, or the MT is rescued at CEN3 (rescue at the KT). MT rescue also happens before the plus end reaches CEN3 (rescue distal to the KT). Once end-on pulling starts, no MT rescue occurs.(C) Time-lapse images of PMET3-CDC20 PGAL-CEN3-tetOs TetR-GFP GFP-TUB1 (T7448) cells. The cells were treated with α factor in methionine drop-out medium with 2% raffinose for 2.5 hr, and then released to YP medium containing 2% galactose, 2% raffinose, and 2 mM methionine. After 3 hr, cells were suspended in synthetic complete medium containing 2% glucose and 2 mM methionine. Images were collected every 4 s for 20 min, using the GFP channel. Zero time is set arbitrarily for the first panel, in which the cell shape is outlined in white. Scale bar, 1 μm. Each example shows (i) end-on attachment/pulling, (ii) MT rescue at the KT (on CEN3), and (iii) rescue distal to the KT. Graphs show MT length (orange) and CEN3–spindle-pole distance (blue) in the cells.(D) The MT plus ends stay with the KT (on CEN3) for an appreciable time prior to rescue, during which the MT shows pausing. Time-lapse images were acquired as in (C). Graph shows the period of MT pausing (shrinkage <1.0 μm/min and no growth), prior to end-on attachment/pulling (1.5–2.0 μm/min; n = 13), MT rescue at the KT (n = 13), and distal to the KT (n = 7).
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fig1: A Shrinking Microtubule, Whose Lateral Surface Is Associated with a Kinetochore, Shows Rescue before or after Its Plus End Reaches the Kinetochore(A) Experimental system to analyze the initial interaction between a KT (on CEN3) and an individual spindle-pole MT (Tanaka et al., 2005).(B) Schematic diagram showing possible fates of a shrinking MT: when a MT plus end catches up with CEN3 on its lateral side, either end-on attachment/pulling of CEN3 occurs, or the MT is rescued at CEN3 (rescue at the KT). MT rescue also happens before the plus end reaches CEN3 (rescue distal to the KT). Once end-on pulling starts, no MT rescue occurs.(C) Time-lapse images of PMET3-CDC20 PGAL-CEN3-tetOs TetR-GFP GFP-TUB1 (T7448) cells. The cells were treated with α factor in methionine drop-out medium with 2% raffinose for 2.5 hr, and then released to YP medium containing 2% galactose, 2% raffinose, and 2 mM methionine. After 3 hr, cells were suspended in synthetic complete medium containing 2% glucose and 2 mM methionine. Images were collected every 4 s for 20 min, using the GFP channel. Zero time is set arbitrarily for the first panel, in which the cell shape is outlined in white. Scale bar, 1 μm. Each example shows (i) end-on attachment/pulling, (ii) MT rescue at the KT (on CEN3), and (iii) rescue distal to the KT. Graphs show MT length (orange) and CEN3–spindle-pole distance (blue) in the cells.(D) The MT plus ends stay with the KT (on CEN3) for an appreciable time prior to rescue, during which the MT shows pausing. Time-lapse images were acquired as in (C). Graph shows the period of MT pausing (shrinkage <1.0 μm/min and no growth), prior to end-on attachment/pulling (1.5–2.0 μm/min; n = 13), MT rescue at the KT (n = 13), and distal to the KT (n = 7).

Mentions: Lateral and end-on KT-MT attachments (see Introduction) found in physiological conditions (Kitamura et al., 2007) can be recapitulated in an engineered assay system, which allows more detailed observation of individual KT-MT interactions (Tanaka et al., 2005, 2007). We used this assay, in which KT assembly was delayed on a particular centromere (CEN3) by transcription from an adjacently inserted promoter (Figure 1A). This procedure increased the distance between CEN3 and the mitotic spindle. While cells were arrested in metaphase, we reactivated CEN3, leading to KT reassembly and interaction with the lateral surface of a single spindle-pole MT (Tanaka et al., 2005).


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)

A Shrinking Microtubule, Whose Lateral Surface Is Associated with a Kinetochore, Shows Rescue before or after Its Plus End Reaches the Kinetochore(A) Experimental system to analyze the initial interaction between a KT (on CEN3) and an individual spindle-pole MT (Tanaka et al., 2005).(B) Schematic diagram showing possible fates of a shrinking MT: when a MT plus end catches up with CEN3 on its lateral side, either end-on attachment/pulling of CEN3 occurs, or the MT is rescued at CEN3 (rescue at the KT). MT rescue also happens before the plus end reaches CEN3 (rescue distal to the KT). Once end-on pulling starts, no MT rescue occurs.(C) Time-lapse images of PMET3-CDC20 PGAL-CEN3-tetOs TetR-GFP GFP-TUB1 (T7448) cells. The cells were treated with α factor in methionine drop-out medium with 2% raffinose for 2.5 hr, and then released to YP medium containing 2% galactose, 2% raffinose, and 2 mM methionine. After 3 hr, cells were suspended in synthetic complete medium containing 2% glucose and 2 mM methionine. Images were collected every 4 s for 20 min, using the GFP channel. Zero time is set arbitrarily for the first panel, in which the cell shape is outlined in white. Scale bar, 1 μm. Each example shows (i) end-on attachment/pulling, (ii) MT rescue at the KT (on CEN3), and (iii) rescue distal to the KT. Graphs show MT length (orange) and CEN3–spindle-pole distance (blue) in the cells.(D) The MT plus ends stay with the KT (on CEN3) for an appreciable time prior to rescue, during which the MT shows pausing. Time-lapse images were acquired as in (C). Graph shows the period of MT pausing (shrinkage <1.0 μm/min and no growth), prior to end-on attachment/pulling (1.5–2.0 μm/min; n = 13), MT rescue at the KT (n = 13), and distal to the KT (n = 7).
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fig1: A Shrinking Microtubule, Whose Lateral Surface Is Associated with a Kinetochore, Shows Rescue before or after Its Plus End Reaches the Kinetochore(A) Experimental system to analyze the initial interaction between a KT (on CEN3) and an individual spindle-pole MT (Tanaka et al., 2005).(B) Schematic diagram showing possible fates of a shrinking MT: when a MT plus end catches up with CEN3 on its lateral side, either end-on attachment/pulling of CEN3 occurs, or the MT is rescued at CEN3 (rescue at the KT). MT rescue also happens before the plus end reaches CEN3 (rescue distal to the KT). Once end-on pulling starts, no MT rescue occurs.(C) Time-lapse images of PMET3-CDC20 PGAL-CEN3-tetOs TetR-GFP GFP-TUB1 (T7448) cells. The cells were treated with α factor in methionine drop-out medium with 2% raffinose for 2.5 hr, and then released to YP medium containing 2% galactose, 2% raffinose, and 2 mM methionine. After 3 hr, cells were suspended in synthetic complete medium containing 2% glucose and 2 mM methionine. Images were collected every 4 s for 20 min, using the GFP channel. Zero time is set arbitrarily for the first panel, in which the cell shape is outlined in white. Scale bar, 1 μm. Each example shows (i) end-on attachment/pulling, (ii) MT rescue at the KT (on CEN3), and (iii) rescue distal to the KT. Graphs show MT length (orange) and CEN3–spindle-pole distance (blue) in the cells.(D) The MT plus ends stay with the KT (on CEN3) for an appreciable time prior to rescue, during which the MT shows pausing. Time-lapse images were acquired as in (C). Graph shows the period of MT pausing (shrinkage <1.0 μm/min and no growth), prior to end-on attachment/pulling (1.5–2.0 μm/min; n = 13), MT rescue at the KT (n = 13), and distal to the KT (n = 7).
Mentions: Lateral and end-on KT-MT attachments (see Introduction) found in physiological conditions (Kitamura et al., 2007) can be recapitulated in an engineered assay system, which allows more detailed observation of individual KT-MT interactions (Tanaka et al., 2005, 2007). We used this assay, in which KT assembly was delayed on a particular centromere (CEN3) by transcription from an adjacently inserted promoter (Figure 1A). This procedure increased the distance between CEN3 and the mitotic spindle. While cells were arrested in metaphase, we reactivated CEN3, leading to KT reassembly and interaction with the lateral surface of a single spindle-pole MT (Tanaka et al., 2005).

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