Figure 4.
Figure 4. Msd1 and Wdr8 are cargo proteins of Pkl1/Kinesin-14. (A) Schematic representation of full-length Pkl1 (Pkl1FL), an NLS mutant (Pkl1nls), and C-terminal truncation (Pkl1Δm) mutant. (B) The NLS sequence within Pkl1 is required for SPB localization of Msd1. Representative mitotic cells containing Msd1-GFP and Cut12-CFP (SPBs; Bridge et al., 1998) with Pkl1FL-mCherry (top) or Pkl1nls-mCherry (bottom) are shown. Each representative image of interphase (I) and mitosis (M) is presented. The positions of SPBs are indicated with arrowheads. (C) Msd1 interacts with Pkl1 that lacks NLS activity or the motor domain. Pull-down assays were performed by using the GFP-trap system, and extracts were prepared from cells containing Msd1-GFP alone (lanes 1 and 6), Pkl1-mCherry (mCh) alone (lanes 2 and 7), or both in pkl1+ (Pkl1FL, lanes, 3 and 8), nls mutant (Pkl1nls, lanes 4 and 9), or motor-less construct (Pkl1Δm, lanes 5 and 10). Immunoblotting with anti-RFP (top) or anti-GFP antibodies (bottom) is shown against whole cell extracts (WCE; lanes 1–4) and pull-down precipitates (lanes 5–8). FL, full length. (D) SPB localization of Msd1 is dependent on the motor domain of Pkl1. Representative mitotic localization of Msd1-GFP in cells containing Pkl1FL-mCherry (top row) or motor-less Pkl1Δm–mCherry (bottom row) is shown. Quantification of signal intensities of Msd1-GFP or Pkl1-mCherry at the SPB is shown at the bottom. The positions of SPBs are indicated with arrowheads. All p-values were obtained from the two-tailed unpaired Student’s t test. Data are presented as the means ± SD (≥50 cells, n = 3). ****, P < 0.0001. a.u., arbitrary unit. (E) The motor-less Pkl1 mutant is defective in spindle anchoring. Mitotic cells containing GFP-Alp4 (SPBs), mCherry-Atb2 (microtubules [MTs]), and Pkl1FL-mCherry (top row) or Pkl1Δm-mCherry (bottom row) were fixed and imaged. The protruding spindle is indicated with arrowheads. P-value is derived from the two-tailed χ2 test (≥50 cells; ****, P < 0.0001). (F) Schematic illustration of the localization scheme for Msd1, Wdr8, and Pkl1. Pkl1 forms a complex with Msd1 and Wdr8 in the cytoplasm and imports this complex into the nucleus. Upon mitotic entry and spindle formation, this complex is transported along the spindle microtubule toward the SPB, to which Msd1 and Wdr8 are responsible for loading this ternary complex through interaction with the γ-TuC. The minus end of the spindle microtubule is subsequently tethered to the SPB. NE, nuclear envelope. The peripheries of the cell and the nucleus are outlined in the images (dotted and continuous lines, respectively). Bars, 10 µm.

Msd1 and Wdr8 are cargo proteins of Pkl1/Kinesin-14. (A) Schematic representation of full-length Pkl1 (Pkl1FL), an NLS mutant (Pkl1nls), and C-terminal truncation (Pkl1Δm) mutant. (B) The NLS sequence within Pkl1 is required for SPB localization of Msd1. Representative mitotic cells containing Msd1-GFP and Cut12-CFP (SPBs; Bridge et al., 1998) with Pkl1FL-mCherry (top) or Pkl1nls-mCherry (bottom) are shown. Each representative image of interphase (I) and mitosis (M) is presented. The positions of SPBs are indicated with arrowheads. (C) Msd1 interacts with Pkl1 that lacks NLS activity or the motor domain. Pull-down assays were performed by using the GFP-trap system, and extracts were prepared from cells containing Msd1-GFP alone (lanes 1 and 6), Pkl1-mCherry (mCh) alone (lanes 2 and 7), or both in pkl1+ (Pkl1FL, lanes, 3 and 8), nls mutant (Pkl1nls, lanes 4 and 9), or motor-less construct (Pkl1Δm, lanes 5 and 10). Immunoblotting with anti-RFP (top) or anti-GFP antibodies (bottom) is shown against whole cell extracts (WCE; lanes 1–4) and pull-down precipitates (lanes 5–8). FL, full length. (D) SPB localization of Msd1 is dependent on the motor domain of Pkl1. Representative mitotic localization of Msd1-GFP in cells containing Pkl1FL-mCherry (top row) or motor-less Pkl1Δm–mCherry (bottom row) is shown. Quantification of signal intensities of Msd1-GFP or Pkl1-mCherry at the SPB is shown at the bottom. The positions of SPBs are indicated with arrowheads. All p-values were obtained from the two-tailed unpaired Student’s t test. Data are presented as the means ± SD (≥50 cells, n = 3). ****, P < 0.0001. a.u., arbitrary unit. (E) The motor-less Pkl1 mutant is defective in spindle anchoring. Mitotic cells containing GFP-Alp4 (SPBs), mCherry-Atb2 (microtubules [MTs]), and Pkl1FL-mCherry (top row) or Pkl1Δm-mCherry (bottom row) were fixed and imaged. The protruding spindle is indicated with arrowheads. P-value is derived from the two-tailed χ2 test (≥50 cells; ****, P < 0.0001). (F) Schematic illustration of the localization scheme for Msd1, Wdr8, and Pkl1. Pkl1 forms a complex with Msd1 and Wdr8 in the cytoplasm and imports this complex into the nucleus. Upon mitotic entry and spindle formation, this complex is transported along the spindle microtubule toward the SPB, to which Msd1 and Wdr8 are responsible for loading this ternary complex through interaction with the γ-TuC. The minus end of the spindle microtubule is subsequently tethered to the SPB. NE, nuclear envelope. The peripheries of the cell and the nucleus are outlined in the images (dotted and continuous lines, respectively). Bars, 10 µm.

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