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First published online 6 June 2006
doi: 10.1242/jcs.03005

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Tuba stimulates intracellular N-WASP-dependent actin assembly

¹ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
² Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA

View larger version (70K): [in a new window]   Fig. 1. mTuba overexpression stimulates dorsal ruffling. (A) Tuba isoforms. Domain structure of full-length Tuba (Tuba) and mini-Tuba (mTuba) are indicated. Tuba possesses four tandem N-terminal SH3 domains (gray boxes), which bind directly to dynamin. The C-terminal SH3 domain binds N-WASP. Schematic representations of mTuba mutants are indicated. (B) Overexpression of GFP-mTuba. B16 cells were transfected with GFP-mTuba and cell lysates were probed for GFP and Tuba using anti-GFP antibody (-GFP) and affinity-purified anti-Tuba antibody (AP -Tuba), respectively. (C) GFP-mTuba expression wave front tracking. The front of GFP-mTuba puncta was tracked through 30 frames of a time-lapse movie and shows apparent random movement. (D) A single image from a time-lapse movie of B16 cells co-transfected with Cherry-mTuba and GFP. The lower panel represents kymographs constructed from the boxed region from each image. Note movement of mTuba puncta but not GFP. (E) Series of time-lapse images of a B16-F1 cell co-transfected with GFP-mTuba and mRFP-actin (Movie 1, supplementary material). Tuba puncta localize to the trailing edge of the actin ruffle and radiate outwards concomitant with the passage of the ruffle. The rectangle indicates the region of the movie used to generate the kymographs. Average speed of ruffles is 1.32±0.38 microns/minute (n=29). Arrows indicate the leading edge of the ruffle. Arrowheads indicate localization of mTuba puncta to the trailing edge of the ruffle. (F) Percentage of total area of Tuba (green) and actin (red) from (E) that overlap during ruffle formation over time (see Materials and Methods). Plot indicates that approximately 10% of the total Tuba population overlaps actin directly, and that approximately 10% of the actin population overlaps Tuba puncta directly. Bars: D, 5 µm; E, 3 µm.

View larger version (96K): [in a new window]   Fig. 2. Inhibition of actin polymerization and deletion of mTuba domains disrupts dorsal ruffle formation. (A) Co-expression of Cherry-SH3 and GFP-actin in B16 cells. Deletion of the actin-regulatory protein-binding domain of mTuba results in cytoplasmic distribution of mTuba with no visible puncta formation. Actin organization is also disrupted. Box indicates region of movie used to generate kymographs. Kymographs confirm cytoplasmic distribution of the mutant. (B) Co-expression of Cherry-GEF and GFP-actin in B16 cells. Deletion of the GEF domain of mTuba results in puncta formation but no ruffling. Box indicates region of movie used to generate kymographs. Kymographs show near-stationary puncta. (C) Co-expression of Cherry-BAR and GFP-actin in B16 cells. Deletion of the BAR domain of mTuba causes extensive peripheral ruffling but no dorsal ruffling. Box indicates region of movie used to generate kymographs. Kymographs show rapid peripheral ruffling activity. (D) B16-F1 cells co-transfected with GFP-mTuba and mRFP-actin and treated with 50 nM cytochalasin D (CD). Image shown is following CD treatment. Boxed area indicates region used to generate kymographs. Average speed of puncta following CD treatment is 0.03±0.19 microns/minute (n=6). Actin polymerization is inhibited, but mTuba remains associated with the actin cytoskeleton at `patches'. Kymographs are derived from the full movie with CD addition at five minutes. Dotted line indicates time of CD addition. Note that, following CD treatment, mTuba puncta fail to move. (E) Percentage of total area of mTuba (green) and actin (red) that coexist during ruffle formation in (D) (see Materials and Methods). Plots show that the percentage of actin overlapping with mTuba puncta increases following CD treatment at actin `patches'. Bars, 5 µm.

View larger version (84K): [in a new window]   Fig. 3. Cdc42 and Arp3 localize to mTuba-stimulated dorsal ruffles. (A) A single frame taken from a 10 minute movie of B16 cells co-transfected with Cherry-mTuba and GFP-Cdc42 shows co-localization of mTuba and Cdc42 at puncta. Box indicates region of movie used for kymograph construction. Average speed of puncta is 1.52±0.34 microns/minute (n=5). (B) Percentage of total area of mTuba (green) and Cdc42 (red) that coexist during ruffle formation in A. 10% of mTuba co-localizes with Cdc42, and 5-10% of the population of Cdc42 co-localizes with mTuba. (C) B16 cells were co-transfected with Cherry-mTuba and GFP-N17Cdc42. N17Cdc42 generates greater numbers of mTuba puncta relative to cells transfected with Cherry-mTuba alone (arrow indicates formation of ruffle). Boxes indicate regions of movie used for kymograph construction. Note limited mobility of mTuba puncta when co-expressed with GFP-N17Cdc42. (D) A single frame taken from a movie of B16 cells co-transfected with Cherry-mTuba and GFP-Arp3 shows co-localization of Arp2/3 and mTuba at puncta. Box indicates region of movie used for kymograph construction. Speed of puncta is 1.01±0.15 microns/minute (n=6). (E) Percentage of total area of mTuba (green) and Arp3 (red) that coexist during ruffle formation in (D). Approximately 90% of mTuba co-localizes with Arp3, and 85% of the population of Arp3 co-localizes with mTuba. Bars: A,D, 5 µm; C, 15 µm.

View larger version (94K): [in a new window]   Fig. 4. mTuba co-localizes with N-WASP, WIP and Toca-1, but not Evl at puncta. (A) B16 cells were co-transfected with GFP-mTuba and mRFP-Evl. Evl localized in discrete bands at the leading edge, distinct from puncta, in mTuba-stimulated dorsal ruffles. (B) B16 cells transfected with GFP-mTuba were cultured on glass coverslips, fixed and stained for N-WASP. Arrowheads indicate robust mTuba and N-WASP co-localization at puncta. (C,D) B16 cells were co-transfected with Cherry-mTuba and either (C) GFP-WIP or (D) GFP-Toca-1. Merged images depict co-localization of mTuba with WIP and Toca-1. Bars: A,C, 3 µm; D, 5 µm; B, 15 µm.

View larger version (22K): [in a new window]   Fig. 5. N-WASP activity is essential for mTuba ruffling. (A) B16 cells were transfected with GFP-mTuba and treated with 10 µM wiskostatin. Images depict mTuba puncta following addition of drug. Wiskostatin abolishes dorsal ruffling and disperses mTuba puncta. (B) Graphical representation of the time-lapse movie from A. Images were thresholded to include the puncta, and the number of thresholded pixels were plotted as a function of time. The intensity of mTuba puncta is reduced by 50% after five minutes treatment and has completely dispersed 8 minutes after addition of the drug (n=3). Bar, 10 µm.

View larger version (59K): [in a new window]   Fig. 6. B16 cells were co-transfected with myc-PIP5K, GFP-mTuba and mRFP-actin. Cells were cultured on glass coverslips and fixed. (A) GFP-mTuba incorporates into comet tails and concentrates at the vesicles. Box indicates region examined in lower panels. (B) PIP5K expression often generated long-tailed comets with mTuba concentrated at the vesicle. Box indicates region examined in lower panels. (C) Schematic representation of targeted sites for RNAi. `A' targets a region in Tuba between the GEF and BAR domains. `B' targets a region between the two C-terminal SH3 domains. (D) B16 cells transfected with control plasmid, RNAi `A' or RNAi `B' were FACS sorted for equivalent expression (50% intensity and above) at 48 hours and lysed 72 hours post-transfection. Cell lysates were subjected to SDS-PAGE and probed with Tuba antibody (Tuba #5415) and ß-actin antibody (loading control). Both RNAi constructs resulted in 75% knockdown of both Tuba and mTuba. (E) B16 cells were transfected with either pRK5myc-PIP5K alone (PIP5K), pRK5myc-PIP5K and Cherry-mTuba (PIP5K + mTuba), pRK5myc-PIP5K and RNAi `A' (PIP5K + RNAi A), or pRK5myc-PIP5K and RNAi `B' (PIP5K + RNAi B). Cells were cultured on glass coverslips and fixed. The number of comets formed per cell was quantitated. RNAi `B' significantly reduced the number of comets observed per cell relative to cells transfected with PIP5K alone. Error bars represent s.d. (***P<0.005, **P<0.01). Bars: A,B, 5 µm.

View larger version (41K): [in a new window]   Fig. 7. Tuba RNAi knockdown inhibits invasive behavior of B16 cells. (A) Matrigel invasion assays of B16 cells transfected with the indicated vectors. Error bars represent s.d. (***P<0.005, **P<0.01). (B) Representative images of the PET membranes from the bottom of Matrigel invasion chambers. Cell area was calculated using Metamorph software and corrected for pore area (see Materials and Methods).

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