Vitre B et al. (2014), Kinetochore-microtubule attachment throughout m...

XB-ART-50476

Mol Biol Cell 2014 Aug 01;2515:2272-81. doi: 10.1091/mbc.E14-01-0698.

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Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E.

Vitre B , Gudimchuk N , Borda R , Kim Y , Heuser JE , Cleveland DW , Grishchuk EL .

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Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore-microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E-dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of "Bonsai" CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore-microtubule attachments during chromosome congression and segregation.

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Species referenced: Xenopus laevis
Genes referenced: bub1b cenpe dld dnai1 myc noct picalml

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	FIGURE 1. In vitro characterization of Bonsai CENP-E functions. (A) Schematics and electron micrographs of individual full-length CENP-E molecules. Our analysis of electron micrographs shows that 3.6% of CENP-E molecules (total N = 84) appear folded. Scale bar, 100 nm. (B) Schematic of full-length (FL), Bonsai (BS), and truncated (TR) CENP-E constructs. C-terminal tail of CENP-E has both a microtubule (MT)-binding site and a motor-binding domain (not shown), which can bind to the motor domain, inhibiting its activity. (C) Representative images of microtubule fields for different CENP-E coverslips. Colored overlays compare microtubules images at different times: red, initial positions; green, 2 s later. Scale bar, 5 μm. (D) Velocity and percentage of moving microtubules for truncated and Bonsai CENP-E. Bars, median ± SEM. At least five independent microscopy chambers were analyzed with truncated and two chambers with Bonsai CENP-E; 10–45 microtubules were analyzed for each chamber. (E) Schematic and montage of stills from a time-lapse of Bonsai CENP-E–coated bead visualized with DIC. Numbers is time in seconds from the start of the bead's motion. Diagram shows velocity of beads coated with full-length, Bonsai, and truncated CENP-E. Bars, mean ± SEM; based on 26 beads for full-length, 20 for Bonsai, and 57 for truncated CENP-E proteins. Data for truncated and full-length CENP-E are from Gudimchuk et al. (2013). (F) Representative kymographs for truncated and Bonsai CENP-E molecules moving on Taxol-stabilized microtubules recorded under identical experimental conditions. Yellow arrowheads point to less bright complexes, which, based on quantitative analysis of fluorescence intensity, represent one or two CENP-E dimers. In Bonsai CENP-E chambers, only larger molecular clusters show directed motility (pink arrows), presumably because occasionally such protein clusters contain uninhibited CENP-E motor heads. (G) Microtubule affinity of truncated, Bonsai, and full-length CENP-E measured in a fluorescence-based pelleting assay with AMPPNP. Mean ± SEM, based on two or more independent experiments. Kd values are reported with 95% confidence.
	FIGURE 2. Analysis of Bonsai CENP-E functions in prometaphase. (A) Schematic of the full-length and Bonsai Myc-Lap-GFP–tagged CENP-E proteins used in DLD-1 cells. (B) Schematic of experimental timeline to prepare cells for endogenous CENP-E depletion, Bonsai or full-length CENP-E rescue and imaging. (C) Representative images of two cells depleted of endogenous CENP-E and expressing full-length or Bonsai CENP-E in the presence of DMSO or CENP-E inhibitor GSK 923295. Green arrowheads point to CENP-E accumulated at the spindle poles. Scale bar, 5 μm. (D) Representative image of a cell with depleted endogenous CENP-E and expressing the Bonsai CENP-E; note the presence of polar chromosomes. Graph shows the average percentage of cells with more than five polar chromosomes. Here and elsewhere, “E” is a control (empty) condition when cells were not rescued. Error bars, SEM; *p < 0.01; p < 0.05; p value of unpaired t test realized on the mean of two (empty) or three (full-length and Bonsai) independent experiments; N = 69 cells for empty, 158 for Bonsai, and 92 for full-length CENP-E. Scale bar, 5 μm. (E) Representative image of BubR1 immunostaining of DLD-1 cells, which were depleted of endogenous CENP-E and induced to express the Bonsai or full-length CENP-E protein. Graph representing BubR1 signal intensity at the kinetochore of unaligned chromosomes in different cells. N = 78 unaligned kinetochores were measured for full-length CENP-E (from 10 cells), 87 for Bonsai (14 cells), and 53 for empty (6 cells). Scale bar, 5 μm.
	FIGURE 3. Reduced stability of kinetochore–microtubule attachment in metaphase in cells expressing Bonsai CENP-E. (A) Representative images of mitotic cells arrested at metaphase with proteasome inhibitor MG132 and subjected to cold for 20 min. Cells were depleted of endogenous CENP-E and rescued with the Bonsai or full-length CENP-E or not rescued. Yellow frame indicates area where tubulin intensity was measured. Scale bar, 4 μm. Right, mean intensity of tubulin signal for cells submitted to cold; p < 0.05, p < 0.01; p value of unpaired t test realized on the mean of three independent experiments. N = 78 cells were analyzed for full-length, 107 for Bonsai, and 70 for empty condition. (B) Representative image used for interkinetochore distance measurement. Purple double-arrowhead indicates the interkinetochore distance. Scale bar, 5 μm. Diagram represents the mean interkinetochore distance in cells depleted of endogenous CENP-E expressing full-length and Bonsai CENP-E. p < 0.05, p value of unpaired t test realized on the mean of two independent experiments. We analyzed 248 kinetochores from 9 cells for full length and 272 from 10 cells for Bonsai. (C) Schematic illustrating timing of cell plating and drug treatment used for experiments in D. Cells were arrested at metaphase with MG132 and imaged live. Noc, nocodazole; Dox, doxycycline. (D) Still images from Supplemental Videos S1 (Full Length) and S2 (Bonsai) showing loss of chromosome alignment (yellow arrowheads) after the chromosomes congressed to the spindle equator. Numbers are time in minutes; scale bar, 10 μm. Right, average time from nocodazole release to first chromosome unalignment event in cells treated as in C. Bars represent the mean time to the first unalignment of chromosome. N = 78 and 58 cells for full-length and Bonsai CENP-E, respectively, based on three independent experiments; N = 33 cells for empty condition based on two independent experiments; ***p < 0.001; p value of an unpaired t test realized on the mean of three independent experiments. Error bars, SEM.
	FIGURE 4. Reduced stability of kinetochore–microtubule attachment in anaphase in cells expressing Bonsai CENP-E. (A) Schematic of the cell plating, RNA interference treatment, and drug treatment for the microtubule destabilization assay. (B) Schematic of the microtubule destabilization assay to test kinetochore attachments. The schematic also presents the outcome of the experiment. (C) Representative image of cells rescued with full-length or Bonsai CENP-E, then tested for stability of kinetochore attachments to depolymerizing microtubule ends as in B. Scale bar, 5 μm. Right, percentage of cells with attached chromosomes without microtubule depolymerization and percentage of cells with unattached outlying chromosomes after 20 min of microtubule depolymerization. Error bars, SEM. *p < 0.01, p value of a unpaired Student's t test realized on the mean of four (Full Length and Empty) or three (Bonsai) independent experiments. For the 20 min depolymerization condition, N = 98 cells were analyzed for full-length, 77 for Bonsai, and 97 for Empty conditions. Anti–centromere antibody (ACA) was used to identify centromeres/kinetochores. (D) Representative images from the time-lapse sequence of a cell with a lagging chromosome (yellow arrowhead). Scale bar, 10 μm. Right, quantification of anaphase cells with lagging chromosomes. Cells were depleted of endogenous CENP-E and rescued with full-length or Bonsai CENP-E or not rescued (Empty); p < 0.05, p value of a unpaired Student's t test realized on the mean of three independent experiments. N = 130 cells were analyzed for full-length, 58 for Bonsai, and 97 for Empty conditions.
	FIGURE 5. Model for the role of CENP-E stalk during mitotic progression. This schematic emphasizes a proposed role of elongated CENP-E stalk during prometaphase in facilitating a microtubule capture by the kinetochores of polar chromosomes, thereby helping to initiate chromosome congression. Our experiments in vitro demonstrate that CENP-E coiled-coil stalk is a positive regulator of CENP-E interactions with microtubules, which may explain why Bonsai CENP-E is not fully active in vivo and induces marked reduction in the stability of kinetochore–microtubule attachments during both metaphase and anaphase. See the text for an extended discussion of other possible molecular mechanisms for the role of CENP-E stalk.

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	FIGURE 1. In vitro characterization of Bonsai CENP-E functions. (A) Schematics and electron micrographs of individual full-length CENP-E molecules. Our analysis of electron micrographs shows that 3.6% of CENP-E molecules (total N = 84) appear folded. Scale bar, 100 nm. (B) Schematic of full-length (FL), Bonsai (BS), and truncated (TR) CENP-E constructs. C-terminal tail of CENP-E has both a microtubule (MT)-binding site and a motor-binding domain (not shown), which can bind to the motor domain, inhibiting its activity. (C) Representative images of microtubule fields for different CENP-E coverslips. Colored overlays compare microtubules images at different times: red, initial positions; green, 2 s later. Scale bar, 5 μm. (D) Velocity and percentage of moving microtubules for truncated and Bonsai CENP-E. Bars, median ± SEM. At least five independent microscopy chambers were analyzed with truncated and two chambers with Bonsai CENP-E; 10–45 microtubules were analyzed for each chamber. (E) Schematic and montage of stills from a time-lapse of Bonsai CENP-E–coated bead visualized with DIC. Numbers is time in seconds from the start of the bead's motion. Diagram shows velocity of beads coated with full-length, Bonsai, and truncated CENP-E. Bars, mean ± SEM; based on 26 beads for full-length, 20 for Bonsai, and 57 for truncated CENP-E proteins. Data for truncated and full-length CENP-E are from Gudimchuk et al. (2013). (F) Representative kymographs for truncated and Bonsai CENP-E molecules moving on Taxol-stabilized microtubules recorded under identical experimental conditions. Yellow arrowheads point to less bright complexes, which, based on quantitative analysis of fluorescence intensity, represent one or two CENP-E dimers. In Bonsai CENP-E chambers, only larger molecular clusters show directed motility (pink arrows), presumably because occasionally such protein clusters contain uninhibited CENP-E motor heads. (G) Microtubule affinity of truncated, Bonsai, and full-length CENP-E measured in a fluorescence-based pelleting assay with AMPPNP. Mean ± SEM, based on two or more independent experiments. Kd values are reported with 95% confidence.
	FIGURE 2. Analysis of Bonsai CENP-E functions in prometaphase. (A) Schematic of the full-length and Bonsai Myc-Lap-GFP–tagged CENP-E proteins used in DLD-1 cells. (B) Schematic of experimental timeline to prepare cells for endogenous CENP-E depletion, Bonsai or full-length CENP-E rescue and imaging. (C) Representative images of two cells depleted of endogenous CENP-E and expressing full-length or Bonsai CENP-E in the presence of DMSO or CENP-E inhibitor GSK 923295. Green arrowheads point to CENP-E accumulated at the spindle poles. Scale bar, 5 μm. (D) Representative image of a cell with depleted endogenous CENP-E and expressing the Bonsai CENP-E; note the presence of polar chromosomes. Graph shows the average percentage of cells with more than five polar chromosomes. Here and elsewhere, “E” is a control (empty) condition when cells were not rescued. Error bars, SEM; *p < 0.01; p < 0.05; p value of unpaired t test realized on the mean of two (empty) or three (full-length and Bonsai) independent experiments; N = 69 cells for empty, 158 for Bonsai, and 92 for full-length CENP-E. Scale bar, 5 μm. (E) Representative image of BubR1 immunostaining of DLD-1 cells, which were depleted of endogenous CENP-E and induced to express the Bonsai or full-length CENP-E protein. Graph representing BubR1 signal intensity at the kinetochore of unaligned chromosomes in different cells. N = 78 unaligned kinetochores were measured for full-length CENP-E (from 10 cells), 87 for Bonsai (14 cells), and 53 for empty (6 cells). Scale bar, 5 μm.
	FIGURE 3. Reduced stability of kinetochore–microtubule attachment in metaphase in cells expressing Bonsai CENP-E. (A) Representative images of mitotic cells arrested at metaphase with proteasome inhibitor MG132 and subjected to cold for 20 min. Cells were depleted of endogenous CENP-E and rescued with the Bonsai or full-length CENP-E or not rescued. Yellow frame indicates area where tubulin intensity was measured. Scale bar, 4 μm. Right, mean intensity of tubulin signal for cells submitted to cold; p < 0.05, p < 0.01; p value of unpaired t test realized on the mean of three independent experiments. N = 78 cells were analyzed for full-length, 107 for Bonsai, and 70 for empty condition. (B) Representative image used for interkinetochore distance measurement. Purple double-arrowhead indicates the interkinetochore distance. Scale bar, 5 μm. Diagram represents the mean interkinetochore distance in cells depleted of endogenous CENP-E expressing full-length and Bonsai CENP-E. p < 0.05, p value of unpaired t test realized on the mean of two independent experiments. We analyzed 248 kinetochores from 9 cells for full length and 272 from 10 cells for Bonsai. (C) Schematic illustrating timing of cell plating and drug treatment used for experiments in D. Cells were arrested at metaphase with MG132 and imaged live. Noc, nocodazole; Dox, doxycycline. (D) Still images from Supplemental Videos S1 (Full Length) and S2 (Bonsai) showing loss of chromosome alignment (yellow arrowheads) after the chromosomes congressed to the spindle equator. Numbers are time in minutes; scale bar, 10 μm. Right, average time from nocodazole release to first chromosome unalignment event in cells treated as in C. Bars represent the mean time to the first unalignment of chromosome. N = 78 and 58 cells for full-length and Bonsai CENP-E, respectively, based on three independent experiments; N = 33 cells for empty condition based on two independent experiments; ***p < 0.001; p value of an unpaired t test realized on the mean of three independent experiments. Error bars, SEM.
	FIGURE 4. Reduced stability of kinetochore–microtubule attachment in anaphase in cells expressing Bonsai CENP-E. (A) Schematic of the cell plating, RNA interference treatment, and drug treatment for the microtubule destabilization assay. (B) Schematic of the microtubule destabilization assay to test kinetochore attachments. The schematic also presents the outcome of the experiment. (C) Representative image of cells rescued with full-length or Bonsai CENP-E, then tested for stability of kinetochore attachments to depolymerizing microtubule ends as in B. Scale bar, 5 μm. Right, percentage of cells with attached chromosomes without microtubule depolymerization and percentage of cells with unattached outlying chromosomes after 20 min of microtubule depolymerization. Error bars, SEM. *p < 0.01, p value of a unpaired Student's t test realized on the mean of four (Full Length and Empty) or three (Bonsai) independent experiments. For the 20 min depolymerization condition, N = 98 cells were analyzed for full-length, 77 for Bonsai, and 97 for Empty conditions. Anti–centromere antibody (ACA) was used to identify centromeres/kinetochores. (D) Representative images from the time-lapse sequence of a cell with a lagging chromosome (yellow arrowhead). Scale bar, 10 μm. Right, quantification of anaphase cells with lagging chromosomes. Cells were depleted of endogenous CENP-E and rescued with full-length or Bonsai CENP-E or not rescued (Empty); p < 0.05, p value of a unpaired Student's t test realized on the mean of three independent experiments. N = 130 cells were analyzed for full-length, 58 for Bonsai, and 97 for Empty conditions.
	FIGURE 5. Model for the role of CENP-E stalk during mitotic progression. This schematic emphasizes a proposed role of elongated CENP-E stalk during prometaphase in facilitating a microtubule capture by the kinetochores of polar chromosomes, thereby helping to initiate chromosome congression. Our experiments in vitro demonstrate that CENP-E coiled-coil stalk is a positive regulator of CENP-E interactions with microtubules, which may explain why Bonsai CENP-E is not fully active in vivo and induces marked reduction in the stability of kinetochore–microtubule attachments during both metaphase and anaphase. See the text for an extended discussion of other possible molecular mechanisms for the role of CENP-E stalk.