Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein

Abstract

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains elusive. Herein, overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end-directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end-directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain.

Figure 1.

Biochemical properties of Nudel mutants. (A) Diagram of Nudel and its mutants used in this work. Numbers indicate positions of amino acid residues. (B) Association of Nudel or mutants with other cellular proteins. FLAG-tagged Nudel or mutants were expressed in HEK293T cells and subjected to coimmunoprecipitation using anti-FLAG M2 resin. Cells transfected with the vector served as a control. Antibodies against the indicated proteins were used for immunoblotting (lanes 6–10). Protein levels in each cell lysate are also shown (lanes 1–5). (C) Nudel^N20 is able to dimerize. GFP-Nudel^P2N was coexpressed with FLAG-Nudel or Nudel^N20 transiently in HEK293T cells (lanes 1 and 2). Control cells expressed only GFP-Nudel^P2N but were cotransfected with the vector pUHD30F (lane 3). After coimmunoprecipitation with anti-FLAG M2 resin (lanes 4–6), samples were immunoblotted with either anti-FLAG mAb (top) or anti-GFP antibody (bottom). The slower migrating form of Nudel^N20 was probably due to phosphorylation (Yan et al., 2003).

Figure 2.

Fragmentation/dispersion of the cis-Golgi cisternae, lysosomes, and endosomes by mutant Nudel. (A, C, and D) CV1 cells transiently expressing Nudel or mutants (green) were processed to show nuclear DNA (blue) and different membrane organelles (red). Transfectants are indicated by arrows. (A) The cis-Golgi cisternae, which was decorated with anti-GM130 antibody (red) after fixation in methanol. FLAG-Nudel isoforms were labeled with anti-FLAG mAb (green). Bar, 15 μm. (B) Statistic results (mean ± SD) showing severity of vesicle dispersion. n = 300, two experiments. (C and D) Lysosomes and transferrin receptor-containing endosomes (red). Vesicles were labeled in living cells before fixation in PFA. GFP-Nudel or mutants were expressed to facilitate identification of the transfectants. Bars, 20 μm.

Figure 3.

Peripheral distribution of endosomes containing WGA-binding sites by Nudel mutant or dynamitin in CV1 cells. (A, B, and D–F) Large arrows indicate transfectants and concave arrowheads indicate vesicles accumulated at the cell processes. Bars, 20 μm. (A and B) Cells expressing FLAG-tagged Nudel^C36 or dynamitin were fixed in methanol and labeled with TRITC-WGA (red), anti-FLAG mAb (green), and DAPI (blue). (C) Statistic results (mean ± SD) showing severity of vesicle dispersion. n = 300, three experiments. (D) Distributions of VSVG-GFP (green) and WGA-positive vesicles (red) in a typical FLAG-Nudel^C36 expressor (blue). Small arrows and arrowheads indicate the Golgi cisternae and typical secretory vesicles, respectively. (E and F) Binding of WGA (red) to the plasma membrane at 4°C and its endocytosis into GFP-Nudel^C36 expressors (green) after 60 min at 37°C.

Figure 4.

Distributions of the ERGIC, COPI-coated compartments, and ER. CV1 cells expressing GFP-tagged (A and B) or FLAG-tagged (D) Nudel isoforms are indicated by arrows. Bars, 20 μm. (A and B) Cells were labeled for the ERGIC with anti-ERGIC53 mAb or COPI-coated vesicles with anti-βCOP mAb. (C) Statistic results (mean ± SD) showing severity of vesicle dispersion. n = 300, two experiments. (D) Cells were labeled for the ER with Texas red–conjugated Con A.

Figure 5.

Impairment of inward vesicle trafficking by the DHC-binding defective Nudel mutant. (A and B) Redistributions of WGA-positive vesicles (red) after nocodazole treatment (Noc) or after recovery from the treatment for 1 h (Rec). CV1 cells expressing Nudel or Nudel^C36 (green) are indicated by arrows. The trans-Golgi cisternae/endosomes (red) and nuclear DNA (blue) were labeled with TRITC-WGA and DAPI. Arrowheads indicate vesicles accumulated at the cell processes. Bar, 20 μm. (C and D) Time-lapse microscopy. Living CV1 cells expressing GFP-Nudel or Nudel^C36 were labeled for lysosomes with LysoTracker and recorded live every 2 s for 120-s consecutive frames showing directional movement of a typical lysosome (arrowheads) and are presented for each type of transfectant. The first and last frames show the GFP fluorescence and tracks of five lysosomes with the longest run lengths, respectively. Red tracks refer to motions with net outward displacements. Where lysosomes cluster is labeled by a star. Track 3 in C corresponds to the indicated inwardly moving lysosome, whereas track 4 in D corresponds to the outwardly moving one. Complete sets of images are available as supplemental material (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200308058/DC1). Bars, 10 μm. (E) Distributions of lysosomes with indicated ranges of total run lengths. (F) Average events of directional motions per cell. See Materials and methods for definitions.

Figure 6.

Inhibition of constitutive protein secretion by mutant Nudel. (A) Secretion of hPL-GFP, but not GFP, from HEK293T cells. (B) Controlled expression of Nudel by tetracycline (Tet). To examine the effects of FLAG-Nudel isoforms on hPL-GFP secretion, HEK293T cells were transfected and cultured in the presence (+) or absence (−) of Tet as described in Materials and methods. At the end of the assays, cell lysates were subjected to immunoblotting using anti-FLAG mAb (top), while β-actin served as a loading control (bottom). (C) BSA as the loading control of culture media. 5 μl of each medium collected at the indicated time points was subjected to Coomassie blue staining after SDS-PAGE. (D) Secretion levels of hPL-GFP. 10 μl of each medium was immunoblotted using anti-GFP antibody. (E) The relative secretion levels at 24 h were quantified for comparison. The relative secretion levels in Tet⁻ samples are normalized to that of corresponding Tet⁺ samples (i.e., 100) and presented as the mean ± SD.

Figure 7.

Golgi fragmentation upon silencing of Nudel in HEK293T cells and a model for Nudel functions in dynein motor activity. (A) Repressed expression of endogenous Nudel by SiRNA. GFP-F, a membrane-associated GFP variant, served as a transfection marker. (B and C) The SiRNA specifically repressed GFP-Nudel expression. (B) The top panels were immunoblotted using anti-GFP antibody. (C) The red fluorescence protein (DsRed) served as a transfection marker. Bar, 50 μm. (D) Effects of Nudel SiRNA on cis-Golgi organization. Cells transfected as in A were labeled with anti-GM130 antibody to decorate the cis-Golgi cisternae. Arrows indicate GFP-F expressors. Bar, 20 μm. (E) A model summarizing the results. We propose that active dynein motor requires Nudel and its interactions with both Lis1 and DHC. Disrupting either interaction or silencing Nudel expression impairs dynein motor activity. Disrupting both interactions inactivate Nudel, thus generating a null mutant. Homodimerization of Lis1 and Nudel (Sasaki et al., 2000) is not considered. Therefore, the actual situations must be more intricate. Existence of NudE further complicates the situation.