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Results: 7

1.
Figure 7

Figure 7. A model for dual-mode regulation of microtubule dynamics by Kip3. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

1. When microtubules grow, Kip3 walks towards and accumulates on the plus end, which is facilitated by the Kip3-tail. 2. At high concentrations, Kip3 triggers microtubule catastrophe. Microtubules then shrink and the bulk of Kip3 molecules are lost from the plus end. 3. Other Kip3 molecules reach the shrinking plus end. At the lower concentrations of Kip3, the stabilizing effects dominate, slowing the shrinkage rate and increasing the rescue frequency, in a manner that requires the Kip3-tail. Note that Kip3 could be affected by or cooperate with other +TIPs to regulate microtubule dynamics, which is not depicted in this model. Asterisks indicate the history of the same motor.

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
2.
Figure 4

Figure 4. Cellular microtubule stability is sensitive to KIP3 dosage. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) Images of cells expressing one or two copies of Kip3ΔT-LZ-EYFP in strains with one or two copies of kip3ΔT-LZ compared to Kip3-EYFP-expressing controls. Microtubules were labeled with CFP-Tub1. Arrows indicate Kip3/Kip3ΔT-LZ on plus ends.
(B) Diminished localization of Kip3ΔT-LZ to cytoplasmic microtubule plus ends in vivo; a two-fold increase in Kip3ΔT-LZ levels restores microtubule plus end fluorescence intensity to levels comparable with Kip3. N=43, 57, and 61 for Kip3, Kip3ΔT-LZ, and 2× Kip3ΔT-LZ, respectively. Shown is mean ± SEM.
(C) Steady-state protein levels of Kip3-EYFP and Kip3ΔT-LZ-EYFP in cells that contained one or two copies of the corresponding genes. Shown is a western blot using an anti-GFP antibody. Tubulin serves as a loading control.
(D) Doubling the gene dosage of kip3ΔT-LZ results in benomyl sensitivity that is indistinguishable from the wild-type control. The indicated strains were spotted at 10-fold dilutions.
(E) Doubling the gene dosage of KIP3 results in benomyl hyper-sensitivity. The indicated strains were spotted at 10-fold dilutions.

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
3.
Figure 6

Figure 6. The Kip3-tail (CT) binds microtubules and tubulin dimers. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) Co-pelleting of the Kip3-tail with taxol-stabilized microtubules. Input: 2.5 µM Kip3-tail. Western blotting was used to detect the Kip3-tail and tubulin because the bands of Kip3-tail and tubulin overlapped on Coomassie-stained gels after SDS-PAGE when the tubulin input was over 10 µM. Asterisk: Input of microtubules only, showing that the antibody recognizing Kip3-tail did not cross-react with tubulin.
(B) Graph of the percentage of Kip3-tail bound plotted against the concentration of polymerized tubulin. Fitting of the data to a one-site saturation-binding model gave a dissociation constant (Kd) of 5.9 ± 0.6 µM. Each data point is presented as mean ± SEM (N=3).
(C) A protein complex between the Kip3-tail and tubulin. Size exclusion chromatography of tubulin (top), CT (Kip3) (middle), and CT (Kip3) + tubulin (bottom). Column fractions were analyzed by Coomassie-staining after SDS-PAGE. Input: 2.5 µM Kip3 tail, 2 µM tubulin. Fraction volume: 0.325 mL. Fractionation of molecular weight standards for this experiment are as follows [molecular weight (KD) / Fraction # / Ve (mL]: 670/26/8.2; 158/35/11.3; 44/45/14.3; and 17/49/15.9.

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
4.
Figure 2

Figure 2. The Kip3-tail is required for normal kinetochore clustering and spindle disassembly during cell division. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) De-clustered kinetochores in Kip3ΔT-LZ expressing cells. Kinetochores are labeled with Nuf2-GFP (red). Microtubules are labeled with CFP-Tub1 (green). Images of Nuf2 were acquired with a YFP filter set to eliminate cross-channel spillover.
(B) Line scans of Nuf2-GFP fluorescence intensity along pre-anaphase spindles in the indicated strains. Note that Nuf2 concentrates near the two spindle poles in KIP3 cells but is loosely scattered along the spindle in kip3Δ and kip3ΔT-LZ cells. Each data point is presented as mean ± SEM (N=15) and is connected by lines to assist visualization.
(C) Quantification of spindles with anaphase lagging chromosomes. N=131, 145, and 142 for KIP3, kip3Δ, and kip3ΔT-LZ cells, respectively.
(D) Defective spindle disassembly in kip3ΔT-LZ cells, detected as a longer anaphase spindle length prior to spindle breakdown. Time-lapse imaging was adopted to define the maximal spindle length prior to mitotic exit in cells containing CFP-Tub1. The comparable lengths of cell long axis indicate the cell sizes are not significantly different among the three strains. Each data point is presented as mean ± SEM (N=15). The spindle lengths of KIP3 and kip3ΔT-LZ cells prior to spindle break down are significantly different by t-test (P<0.00001).

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
5.
Figure 1

Figure 1. The Kip3-tail is critical for the Kip3-mediated regulation of microtubule stability in vivo. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) Schematic of the constructs used in this study. Kip3 contains an N-terminal motor domain (Head, red), followed by a short coiled-coil domain (blue), and a C-terminal tail domain (orange).
(B) Comparable benomyl resistance of kip3ΔT-LZ and kip3Δ stains relative to the KIP3 control strains. Shown are 10-fold dilutions of cells from top to bottom spotted onto plates. TOP: benomyl-containing plate. BOTTOM: control YPD plate.
(C) Similar steady-state protein levels of Kip3 and Kip3ΔT-LZ expressed from the endogenous KIP3 promoter. Kip3/Kip3ΔT-LZ contains an EYFP tag and was detected with an anti-GFP antibody. Tubulin serves as a loading control.
(D) Deletion of the Kip3-tail does not alter its distribution between the nucleus and the cytoplasm. TOP: single focal plane, wide-field images of Kip3-EYFP or Kip3ΔT-LZ-EYFP (red) expressing cells co-stained with DAPI (blue). Microtubules were depolymerized by treatment of cells with 35 µg/mL nocodazole for 15 min. The bright EYFP dot is the motor associated with spindle pole bodies or kinetochores. Cell boundaries are outlined by dashed while lines. BOTTOM: quantification of the ratio of nuclear to cytoplasmic fluorescence intensity of Kip3-EYFP and Kip3ΔT-LZ –EYFP. Pre-anaphase and anaphase cells were scored for the analysis. The data was represented as mean ± SEM (N=75).

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
6.
Figure 3

Figure 3. The Kip3-tail is required for efficient microtubule plus end binding and microtubule depolymerase activity in vitro. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) Coomassie blue-stained gel with the indicated proteins after purification.
(B) Kymographs from single molecule imaging experiment visualizing the motility of TMR-labeled Kip3 and Kip3ΔT-LZ on Taxol-stabilized microtubules by TIRF microscopy. An image of fluorescein-labeled microtubules was acquired separately to determine the position of microtubule ends, as indicated by white asterisks.
(C) Comparison of the microtubule on-rate, run length, velocity, and plus end dwell time between Kip3 and Kip3ΔT-LZ. The microtubule effective on-rate was defined as the number of processive translocation events (>0.5 µm in run length) observed per min divided by the length of each microtubule and the motor input concentration. The on-rate for thirteen microtubules in each group was averaged. The velocity was fit to a Gaussian curve. The run length and dwell time at the plus ends were fit to a first-order exponential curve. The bar graphs show mean ± SEM. Histograms of velocity, run length, and dwell time are shown in Figure S2.
(D) Depolymerization of microtubules by Kip3 and Kip3ΔT-LZ. Kymographs show depolymerization of GMPCPP-stabilized fluorescein-labeled microtubules. Motor concentrations and estimated flux rates are indicated. Left and middle panels compare microtubule depolymerization rates at the same input concentration of motors; left and right panel compare microtubule depolymerization rates at equal motor flux on plus ends. Images were acquired at 15 sec intervals.
(E) Depolymerization rates compared at either equal motor input (top) or equal flux on plus ends (bottom). Shown is mean ± SEM (N>=10). Microtubules with an initial length of 6 – 8 µm were chosen for analysis.

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.
7.
Figure 5

Figure 5. The Kip3-tail enables the binding of kinesin-1 (KHC560) to microtubule plus ends and promotes microtubule stabilization. From: Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

(A) Kymographs from TIRF imaging visualizing single KHC560 or KHC560-CT (KHC-Kip3 tail fusion) molecules walking on Taxol-stabilized microtubules. An image of fluorescein-labeled microtubules was acquired separately to determine the position of microtubule end, as indicated by while asterisks. Note the long dwell time of KHC560-CT on microtubule ends, seen as vertical streaks on the kymograph.
(B) The run length, velocity and plus end dwell time of KHC560 and KHC560CT. The velocity was fit to a Gaussian curve. The run length and dwell time on plus ends were fit to a first-order exponential curve. Shown is mean ± SEM. Histograms of velocity, run length, and dwell time are shown in Figure S2. Pauses during the translocation of motors were excluded when calculating the velocity.
(C) Comparable steady-state protein levels of KHC560-CT and KHC560. KHC560-CT-EYFP and KHC560-EYFP were detected by western blot using anti-GFP antibody.
(D) Enrichment of KHC560-CT on astral microtubule plus ends. KHC560-EYFP and KHC560-CT-EYFP were expressed from the KIP3 promoter. Microtubules were labeled with CFP-Tub1. Strong enrichment (3 fold over cytoplasmic background) of KHC560-CT is observed on 34% of cytoplasmic microtubule plus ends (arrows) whereas enrichment of KHC560 was observed on 0% of microtubule plus ends (N=207). The average cytoplasmic microtubule length was 2.0 ± 0.1µm (mean ± SEM) in KHC560 – expressing cells whereas it was 4.3 ± 0.2 µm in KHC560-CT- expressing cells. Asterisks indicate long and buckled microtubules. Cell boundaries are outlined by dashed while line.
(E) The Kip3-tail (CT) inhibits microtubule shrinkage in vitro. TOP: Taxol-stabilized, fluorescein-labeled microtubules were induced to undergo shrinkage by removing Taxol and adding 100 mM KCl with or without the Kip3-tail. Shown are microtubules remaining at selected time points after shrinkage was induced. Individual microtubules are color-coded to facilitate visual tracing of shrinkage over time. BOTTOM: quantification of the shrinkage rate. Shown is mean ± SEM (N=50).

Xiaolei Su, et al. Mol Cell. ;43(5):751-763.

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