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doi: 10.1242/10.1242/jcs.00630

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Receptor activation regulates cortical, but not vesicular localization of NDP kinase

Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, VA 22908, USA

View larger version (78K): [in a new window]   Fig. 1. Localization of NDP kinase in quiescent and EGF-treated NIH-3T3 cells. (a) In resting cells, NDP kinase accumulates in ring-like structures (v) that correspond to phase-bright cytoplasmic vesicles (b). (c) In cells treated with 10 ng ml–1 EGF for 2 minutes, NDP kinase is present in lamellipodia and ruffles (r) as well as around cytoplasmic vesicles (v); (d) the corresponding phase-contrast image. Arrowheads indicate areas where the intensity of the fluorescence signal does not coincide with phase-dark areas. NDP kinase was visualized with polyclonal Ab-1. Scale bar, 25 µm.

View larger version (71K): [in a new window]   Fig. 2. The strong signal for NDP kinase around vesicles and in ruffling membranes is not a volume effect. Fluorescence ratio imaging of cells transfected with GFP and stained with anti-NDP kinase-A after treatment with 10 ng ml–1 EGF for 1 minute. (a) NDP kinase A, (b) GFP and (c) corresponding phase-contrast image. (d) Ratio image showing high NDP kinase A/GFP ratios (arbitrarily set as dark areas) at edge ruffles and around large vesicles in the perinuclear area. Scale bar, 25 µm.

View larger version (36K): [in a new window]   Fig. 3. Stimulation of cells with bombesin, a GPCR agonist, also induces translocation of NDP kinase to the actin-rich cell cortex. Serum-starved cells were treated with 10 nM bombesin for 10 minutes, then stained with Ab-1 antibody (a) and rhodamine-phalloidin (b). Arrows show accumulation of NDP kinase around vesicles. The arrowheads indicate patches where NDP kinase staining extends beyond the actin-rich ruffling edge. Scale bar, 25 µm.

View larger version (19K): [in a new window]   Fig. 4. BDM suppresses NDP kinase translocation to the cell cortex. Cells were pre-incubated with 30 mM BDM for 30 minutes as described in Materials and Methods. The incubation was continued in the presence of 10 ng ml–1 EGF for 2 minutes, followed by fixation and labeling with anti-NDP kinase A (a) and rhodamine-phalloidin (b). Notice the absence of NDP kinase staining in cell protrusions that remain enriched in F-actin (b, arrowheads). Scale bar, 50 µm.

View larger version (55K): [in a new window]   Fig. 5. Rac induces recruitment of NDP kinase to ruffles. Cells transfected with HA-tagged RacG12V (a,b,c) or RacT17N (d,e,f) were serum starved. Cells expressing RacG12V were fixed directly, whereas those expressing T17N Rac were first treated with EGF for 2 minutes. Samples were double labeled for the HA tag (a,d) and NDP kinase (b,e) with Ab-1. (c,f) Phase contrast images. In the cell that expresses activated Rac (a), NDP kinase is visible at the edge of the lamellipodium (b, arrow) and around vesicles (b, arrowheads). Expression of T17N Rac (d) inhibits accumulation of NDP kinase in ruffles in response to EGF (e; compare the transfected cell on the left with those on top right) but does not affect vesicular NDP kinase (e, arrowhead). Scale bar, 20 µm.

View larger version (110K): [in a new window]   Fig. 6. Limited overlap of Tiam1 and NDP kinase in ruffles. Serum-starved cells were treated with 10 ng ml–1 EGF for 2 minutes. Cells were stained with anti-NDP kinase A (a) and anti-Tiam1 (b) antibodies; (c) phase contrast image. Notice that NDP kinase is present throughout the ruffle, whereas Tiam1 is confined to a small area (arrow). The merged image (d) highlights the limited overlap between the signals for Tiam1 and for NDP kinase. (c) Scale bar, 25 µm.

View larger version (38K): [in a new window]   Fig. 7. Cell stimulation increases the association of NDP kinase and Rac with cell membranes. (A) Merged image of serum-starved cells treated with EGF for 2 minutes and stained for Rac (red) and NDP kinase (Ab-1; green); areas of overlap appear in yellow. The line scan analysis shows the fluorescence intensities of Rac and NDP kinase along the white line in the image. Scale bar, 25 µm. (B) Subcellular fractionation of quiescent and serum-treated cells was as in Methods. The particulate fraction was immunoblotted with antibodies to NDP kinase (NDPK), Rac and Na+,K+-ATPase, a plasma membrane marker.

View larger version (52K): [in a new window]   Fig. 8. Characterization of the vesicles labeled by NDP kinase. (A). Size distribution of NDP kinase-labeled vesicles. The histogram was built as described in Materials and Methods. (B) NDP kinase is associated with vesicles labeled by LAMP-1. This image shows the immunolocalization of NDP kinase with Ab-1 (red) and LAMP-1 (green) in quiescent cells. NDP kinase labels the periphery (a) of vesicles, whereas LAMP-1 is visible in the lumen (b, arrows); a circular ruffle (arrowhead) is stained only by NDP kinase. Scale bar, 25 µm.A large vacuole positive for both NDP kinase (c) and LAMP-1 (d) has a multivesicular appearance. Scale bar, 5 µm.

View larger version (70K): [in a new window]   Fig. 9. Microtubular association of NDP kinase-labeled vesicles. Cells were fixed and double stained with antibodies to NDP kinase (Ab-1) and -tubulin after being serum starved for 18 hours (–serum), kept in complete medium (+serum) or treated with 33 µM nocodazole for 1 hour (nocodazole). Arrowheads show vesicular structures visible in phase contrast, which are labeled by antibodies to -tubulin and NDP kinase in serum-starved and serum-treated cells (left and middle, respectively). Labeling of vesicles is lost in cells treated with nocodazole (right). Scale bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 10. NDP kinase pellets with microtubules and endosomal vesicles. Prior to homogenization, cells were incubated in the absence and presence of 10 ng ml–1 EGF for 15 minutes. Taxol-stabilized microtubules and associated membranes were then isolated as described in Materials and Methods, in the absence of nucleotides. Fractions were analysed by immunoblotting with antibodies to NDP kinase, tubulin and the endosomal markers Rab4 and LAMP-1.

View larger version (23K): [in a new window]   Fig. 11. Nucleotides release NDP kinase from taxol-stabilized microtubules and associated vesicles. (A) An extract from resting cells was divided into equal portions that were incubated with Taxol in buffer supplemented or not with ATP or GTP (both at 1 mM); after washes, pellets were tested for NDP kinase, tubulin, Rab4 and LAMP-1. (B) As above; binding of NDP kinase to the MT/Ves pellets was assessed in samples where GTP or guanine nucleotide analogs were included in the incubation with taxol.

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