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First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02622

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Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome

Martina Casenghi, Francis A. Barr^*, and Erich A. Nigg^*,

Max-Planck Institute of Biochemistry, Department of Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany

View larger version (65K): [in a new window]   Fig. 1. Effect of Nlp and ninein overexpression on Golgi organisation. (A) Tet-On U2OS stable cell lines carrying Myc-Nlp, Myc-ninein or EGFP-C-Nap1 were either treated with tetracycline (+Tet) or left untreated (–Tet). Cells were then grown for 5 hours, fixed and analysed by immunofluorescence microscopy. Overexpressed proteins were detected with anti-Myc antibodies or using EGFP fluorescence (green). The Golgi was detected using antibodies against the Golgi matrix protein GM130 (red). DNA was stained with DAPI (blue). (B) Expression of Myc-Nlp, Myc-ninein or EGFP-C-Nap1 was either induced in Tet-On U2OS stable cell lines with tetracycline (black bars), or the cells were left untreated (grey bars). Histograms show the percentage of cells with a fragmented staining for the Golgi marker GM130, calculated from three independent experiments in which 400-600 cells were counted. Error bars indicate standard deviations. (C) Myc-tagged Nlp and ninein were expressed in U2OS cells. These were then stained with Myc antibodies to detect the overexpressed protein (green) and counterstained with antibodies against -tubulin (red). Bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 2. Nlp and ninein associate with the dynein-dynactin complex. (A,B) U2OS cells expressing Myc-Nlp, Myc-ninein or EGFP-C-Nap1 were analysed by immunofluorescence microscopy. Overexpressed proteins were detected with anti-Myc antibodies or using EGFP fluorescence (green). Induced (+Tet) and control (–Tet) cells were counterstained with antibodies to (A) anti-p150Glued or (B) the dynein intermediate chain (DIC) (red). DNA was stained with DAPI (blue). Arrowheads indicate centrosomes. Bars, 10 µm. (C) Immunoprecipitation experiments were performed from extracts of HEK293T cells transfected with EGFP-tagged Nlp, ninein or C-Nap1 using anti-GFP antibodies. Immunoprecipitates were analysed by blotting with antibodies against GFP, the dynactin subunits p150Glued and p50-dynamitin, and pericentrin.

View larger version (35K): [in a new window]   Fig. 3. The N-terminal domains of Nlp and ninein bind the dynein-dynactin complex. (A) Whole cell extracts were prepared as described in the Materials and Methods and incubated in the presence of GST-Nlp full length, GST-Nlp N terminus and C terminus, GST-ninein N terminus or GST-GM130cc. The bound fractions were analysed by blotting with antibodies against dynactin subunits. Dynein was detected using antibodies against the dynein intermediate chain (DIC). The amount of total protein extract loaded was 1/80 of the amount of the bound fractions. The left panel shows a representative Coomassie Blue-stained gel to illustrate GST-fusion proteins. (B) Endogenous Nlp was precipitated from HEK293T cell extracts using rabbit polyclonal anti-Nlp antibodies. Rabbit pre-immune antibodies were used as control. Immunoprecipitates were analysed by blotting using antibodies against the dynactin subunits p150Glued, p50-dynamitin and Arp1.

View larger version (61K): [in a new window]   Fig. 4. The dynactin-binding domains of Nlp and ninein are required for the Golgi fragmentation phenotype. (A,B) U2OS cells were transfected with EGFP-tagged Nlp, NlpEF-h I (1-38aa), EGFP-NlpEF-h I-II (1-350aa), and the Nlp N- and C-terminal domains, or the EGFP-tagged N-terminal domain of ninein. Expressed proteins were detected using EGFP fluorescence (green). (A) The Golgi was stained with antibodies against GM130 (red), transfected cells are marked by an asterisk. (B) the dynactin complex was stained with antibodies against p150Glued (red). DNA was stained with DAPI (blue). Bars, 10 µm. (C) A schematic representation of the Nlp and ninein deletion mutants, and a summary of their effects on p150Glued and the Golgi.

View larger version (77K): [in a new window]   Fig. 5. Centrosomal targeting of Nlp and ninein depends on dynein-mediated transport. (A) Tet-On U2OS cell lines carrying Myc-tagged Nlp or ninein were treated with nocodazole (left panels) or DMSO (middle panels) for 1 hour before Nlp and ninein expression was induced for 5 hours by addition of tetracycline. Alternatively, Nlp and ninein expression was induced for 4 hours by addition of tetracycline, followed by a 4 hours treatment with nocodazole (right panels). Cells were fixed and then stained with antibodies to the Myc-epitope to detect Nlp and ninein (green), and -tubulin (red). (B) U2OS cells treated with nocodazole or DMSO for 4 hours prior to fixation were stained with antibodies to Nlp and -tubulin. (C) Tet-On U2OS stable cell lines carrying Myc-tagged Nlp or ninein were transfected with DsRed-p150 CC1. Addition of tetracycline to the culture medium 12 hours after transfection was used to induce expression of Nlp and ninein. Cells were then grown for 12 hours in the presence of tetracycline, fixed and analysed by immunofluorescence microscopy. Cells transfected with p150 CC1 were detected by DsRed fluorescence (asterisks). Cells were counterstained with anti-Myc antibodies and DNA was stained with DAPI (blue). Bars, 10 µm. A schematic representation of the experimental procedures is shown below each set of images.

View larger version (57K): [in a new window]   Fig. 6. Plk1 regulates the association of Nlp with the dynein-dynactin complex. (A) U2OS cells were transfected with EGFP-Nlp or EGFP-Nlp8 (green) together with either Plk1K82R or Plk1T210D, and the localisation of p150Glued (red) was analysed by immunofluorescence microscopy. Bar, 10 µm. (B) Nlp was immune precipitated from extracts of `Tet-On' Myc-Nlp-expressing cells using antibodies to the Myc-epitope; control precipitations were performed using a non-specific IgG fraction. Samples of the cell lysate, unbound, and bound fractions were western blotted for Nlp and p150glued. (C) Total cell extracts were incubated with the GST-Nlp N terminus, GST-Nlp N terminus mock-treated with Plk1 and GST-Nlp N terminus phosphorylated by Plk1. Bound fractions were analysed by immunoblotting using antibodies to p150Glued and Plk1 (left panel), and Coomassie Brilliant Blue staining (right panel).

View larger version (25K): [in a new window]   Fig. 7. Plk1 regulates Nlp transport and attachment to the centrosome. In interphase cells, Nlp is transported to the centrosomes by the dynein-dynactin motor complex. At the centrosome it then contributes to the attachment and nucleation of microtubules via the -tubulin ring complex (-TuRC). In mitosis, Plk1 phosphorylates Nlp and exerts a dual regulatory function. Phosphorylated Nlp is unable to interact with either (i) its binding partners at the centrosome or (ii) the dynein-dynactin motor complex. This dual action of Plk1 both promotes the release of Nlp from the centrosome and prevents its re-supply by the dynein-dynactin motor complex, and as a consequence Nlp redistributes into the cytoplasm during mitosis. Mitosis-specific -TuRC binding proteins (GTBPs) then replace Nlp at the centrosome as part of the centrosome maturation process required for the formation of the mitotic spindle.

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