Nudel modulates kinetochore association and function of cytoplasmic dynein in M phase

Abstract

The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly ( approximately 78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.

Figure 1.

Subcellular distributions of Nudel in HEK293T cells. (A) Localization of Nudel at the outer kinetochore. Mitotic chromosome spreads were prepared and immunostained to visualize the indicated proteins. Chromosome DNA was stained with DAPI. Arrowheads indicate representative kinetochores that are shown in the 4′ enlargements. (B) Distributions of Nudel in the cell cycle. Typical cells in the indicated phases are shown. Arrowheads point to typical kinetochore staining. Arrows indicate centrosomes or spindle poles. Asterisks mark the midbody. Scale bar, 10 μm.

Figure 2.

Nudel still binds kinetochores after p50 overexpression or ZW10 depletion. HEK293T cells were transfected with pEGFP-p50 (A), pTER-Luci (B and C, panels 1–4), or pTER-ZWi (B and C, panels 5–8) and treated with nocodazole before fixation. Arrowheads indicate representative kinetochores. Quantitation results are shown on the right. Total n is listed beside each pair of histograms. Scale bar, 10 μm. (A) Typical mitotic cells overexpressing GFP-p50. Panels 1–4 also contain an untransfected cell as a control. (B and C) Typical control (panels 1–4) or ZW10-depleted (panels 5–8) mitotic cells.

Figure 3.

Mitosin recruits Nudel to the kinetochore. (A) Kinetochore staining of typical cells transfected with pBS/U6 (panels 1–3) or pBS/U6/Mi-1 (panels 4–10). Cells were treated with nocodazole before fixation. Quantitation results for kinetochore intensity are shown on the right. n = 157. (B) Diagrams of Nudel and Nudel^C36. (C) Coimmunoprecipitation of GFP-tagged Nudel, but not Nudel^C36, with FLAG-mitosin. Cells overexpressing the indicated proteins (lanes 2–4) were treated with nocodazole (Noc, 0.4 μg/ml) for 16 h to enrich M phase populations and subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated resin (lanes 5–7). Proteins were separated with 3–12% gradient SDS-PAGE and immunoblotted with anti-GFP (top panel) or anti-FLAG (bottom panel) antibodies. The top band of GFP-tagged Nudel or Nudel^C36 seen in nocodazole-treated cells (arrowhead) was a phosphorylated form. (D) Kinetochore localization of GFP-tagged Nudel but not Nudel^C36. Arrows, spindle poles; arrowheads, typical kinetochores. Scale bar, 10 μm.

Figure 4.

Effects of Nudel depletion on kinetochore dynactin. HEK293T cells were transfected with either pTER (panels 1–4) or pTER-Nudi (panels 5–8) for 72 h. Typical cells either in early prometaphase (A) or after nocodazole treatment (B) are shown. Arrowheads indicate representative kinetochores. Scale bar, 10 μm. (C) Relative kinetochore intensities of the indicated proteins. Total n is shown over each pair of histograms.

Figure 5.

Nudel depletion reduces kinetochore-bound dynein, dynactin, Lis1, and CLIP-170. (A–D) Immunostaining of typical nocodazole-treated cells transfected twice with pTER (panels 1–4) or pTER-Nudi (panels 5–8). Arrowheads, representative kinetochores. Scale bar, 10 μm. (E) Relative kinetochore intensities of the indicated proteins. Total n is shown over each pair of histograms.

Figure 6.

Nudel is crucial for poleward transport of kinetochore proteins. (A and B) Nudel^C36 overexpression or Nudel depletion represses poleward kinetochore protein transport. Mitotic HEK293T cells were subjected to ATP inhibitor assays (Howell et al., 2001) and immunostained for Rod or p150^glued. For convenience, GFP-F was used as a transfection marker for mock-depleted (control) or Nudel-depleted (Nudel RNAi) cells (Liang et al., 2004). A typical late prometaphase cell is shown. (C) Existence of MT-kinetochore attachment in late prometaphase cells overexpressing Nudel^C36 or lacking Nudel. A representative optical section is shown. Insets are 2′ enlargements in which MT staining is enhanced digitally for better views of kinetochore fibers. (D) Mitosin-depletion does not inhibit poleward kinetochore protein transport. Cells were immunostained for mitosin and p150^glued after ATP inhibitor assays. Concaved arrows, spindle poles. Scale bar, 10 μm.

Figure 7.

Effects of Nudel on M phase progression. (A) Representative cell cycle profiles. HEK293T cells expressing GFP-Nudel^C36 or GFP-Nudel^N20/C36 were analyzed by flow cytometry. (B) Summary of time-lapse studies on M phase progression. Mitotic cells were randomly picked and imaged till anaphase onset or for 60 min (Nudel or N20/C36-overexpressing cells)/120 min (N20- or C36-overexpressing cells). Total n is shown over each group. (C) Representative time-lapse images. Early mitotic cells coexpressing H2B-GFP and RFP-Nudel or Nudel^C36 were recorded at 1.5-min intervals for up to 3 h. Arrows indicate metaphase plate. Scale bar, 10 μm. Also see Supplementary Videos 1 and 2. (D) Effect of Nudel depletion on mitosis. Cells were transfected twice with either vector (Vec) or pTER-Nudi (RNAi) as described in Materials and Methods. H2B-GFP was transiently expressed as both transfection and chromosome markers. Cells in early M phase were recorded at 1.5-min intervals for up to 3 h. Also see Supplementary Videos 3 and 4.

Figure 8.

A model for Nudel functions in M phase. Dynein/dynactin binds the kinetochore through the Rod/ZW10/Zwilch complex. Nudel is mainly recruited by mitosin (CENP-F), whereas a portion of it also binds dynein and Lis1 independent of mitosin. Nudel associated with both dynein, and Lis1 activates dynein/dynactin-mediated poleward transport of outer kinetochore proteins, such as ZW10, Rod, and Mad2 (not shown), to facilitate inactivation of the spindle checkpoint. It also activates NuMA transport for spindle assembly. On the other hand, when interacting with both mitosin and dynein, Nudel stabilizes dynein/dynactin against MT-dependent stripping to facilitate the motor's force generation function for chromosome movement and tension. See text for detailed discussion.