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Pax3 regulates morphogenetic cell behavior in vitro coincident with activation of a PCP/non-canonical Wnt-signaling cascade

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada

View larger version (110K): [in a new window]   Fig. 1. Formation of distinct cell arrangements during Pax3-induced aggregation of Saos-2 cells. Representative phase-contrast images depicting the arrangement of Saos-2 cells postinfection at the times indicated, infected with control Ad-ß-gal infected (A-C) or Ad-Pax3flag (D-E) adenoviruses. Beginning at 48 hours postinfection, in contrast to the random arrangement of control infected cells (A-C), many Pax3-infected cells could be found in columnar (oriented parallel to the plane of the substratum) arrangements (D). These columnar arrangements persisted in Pax3-infected cells as they formed aggregates (E-F). Bars, 80 µm.

View larger version (7K): [in a new window]   Fig. 2. Ectopic Pax3 expression induces increased cell motility in Saos-2 cells. Paths of individual cells from representative time-lapse recordings are depicted by dots showing centroid positions. Centroid positions were computed at 10 minute intervals over a 10 hour period, at 36 hour postinfection of control infected (A), or Ad-Pax3flag-infected (B) cells.

View larger version (11K): [in a new window]   Fig. 3. Sequential time-lapse images depicting cell behaviors underlying the formation of a Pax3-induced columnar cell arrangement. Tracings of cells from selected intervals (A,B) revealed that cell rearrangement occurred by means of cell migration and cell intercalation. (B) Tracing of cells in A 10 hours later. Products of cell division are indicated by the letters a and b. The cell labelled N indicates a new cell that migrated into the field.

View larger version (147K): [in a new window]   Fig. 4. Ectopic Pax3 expression in Saos-2 cells induces directed cell protrusive behavior. F-Actin staining of Ad-ß-gal control infected (A, top panel) or Ad-Pax3flag-infected cells (A, bottom panel) at 72 hours postinfection illustrates the presence of multiple stable lamellipodia and filopodia-like protrusions in Ad-Pax3flag-infected cells. The episodic nature of these protrusions is illustrated by the retraction of a lamelliform protrusion (arrowhead) in sequential stills (D-G) from a time-lapse recording of Pax3-infected cells. Pax3-induced cell protrusions were often elaborate, resembling growth cones (B). Stable filopodia-like protrusions from individual cells were frequently observed to be directed at neighboring cells (C and arrow in E). Time-lapse analysis revealed that these protrusions were involved in cell behavior that resulted in fusion of aggregates (D-G) and in recruitment of free cells to aggregates (H-J) (see text for details). Note cell extending from aggregate to neighboring cell (arrow in H). Bars, 20 µm (A); 40 µm (B,C); 50 µm (D); 80 µm (H).

View larger version (109K): [in a new window]   Fig. 5. Subcellular distribution and relocalization of endogenous Dishevelled in Saos-2 cells in response to Pax3 expression. Confocal images of methanol/acetone-fixed Ad-ß-gal-infected (A, middle panel) and Ad-Pax3flag-infected (A, right panel and B) cells, at three days postinfection, and uninfected (A, left panel) cells. Methanol/acetone fixation results in complete extraction of virally encoded GFP (B, left panel), with minimal staining with control FITC- (B, middle panel) or Texas Red-conjugated (B, right panel) secondary antibodies. In control cells Dishevelled staining is punctate and cytoplasmic with prominent staining along stress fibers where Dishevelled colocalizes with that of F-actin (C, top row). In cells expressing Pax3, Dishevelled accumulates at the plasma membrane and at cell-cell junctions where it colocalizes with cortical actin bundles (A, right panel; and C, bottom row). In Pax3-expressing cells, Dishvelled also accumulates peripherally around the nucleus and at the tips of filopodia-like protrusions. Bars, 20 µm.

View larger version (121K): [in a new window]   Fig. 6. Localization of endogenous Frizzled to the actin cytoskeleton and its redistribution in Saos-2 cells expressing Pax3. Confocal images of methanol/acetone-fixed (A-C) and paraformaldehyde-fixed (D-K) Saos-2 cells treated as indicated. In control cells (A,B,D) and cells expressing Pax3flag (C,E), Frizzled localized to the tips of protrusions, at sites resembling focal adhesions, and in a punctate diffuse manner throughout the cytoplasm. In control cells Frizzled signals overlapped with those of F-actin (detected by phalloidin staining) along stress fibers (arrow in F-G), with prominent Frizzled signals at the ends of stress fibers (H). Cells expressing Pax3flag have fewer stress fibers (J) relative to control cells (G), and in paraformaldehyde-fixed Pax3-expressing cells, an accumulation of Frizzled signals to cytoplasmic vesicles is apparent (arrowheads in E and I). Cells in F-K were fixed with a paraformaldehyde-cytoskeletal fixation procedure (see Materials and Methods). Bars, 20 µm.

View larger version (98K): [in a new window]   Fig. 7. Frizzled colocalizes with vinculin and co-immunoprecipitates with vinculin from NP-40 detergent-insoluble cell extracts. Uninfected (A-C), Ad-ß-gal-infected (D-F) and Ad-Pax3flag infected (G-H) paraformaldehyde (cystoskeletal)-fixed cells were dual labeled with anti-Frizzled (A,D,G) and anti-vinculin (B,E,H) antibodies. Confocal images of dual-stained cells shows near perfect overlap of Frizzled with vinculin at focal adhesions in control cells. In cells expressing Pax3 there was a reduction of Frizzled signals at many vinculin-containing focal adhesions (arrow in G and H); in these cells Frizzled signals overlapped extensively with vinculin in cytoplasmic vesicles (arrowhead in G-I). Strong nuclear staining of Frizzled and vinculin appeared to be an artifact of paraformaldehyde-cytoskeletal fixation. The subcellular distribution of Frizzled was examined by western blots of uninfected Saos-2 cell extracts fractionated into NP-40 soluble (S) and cytoskeletal-associated insoluble (I) extracts (J). (K) Frizzled co-immunoprecipitates with vinculin (lanes 7) and vice versa (lane 8) from insoluble extracts but not from soluble extracts (lanes 4 and 5, respectively) of uninfected Saos-2 cells. Dishevelled antibodies co-immunoprecipitate both vinculin and Frizzled from insoluble extracts (lane 9). Lane 1 represents control immunoprecipitation (IP) of insoluble extracts with IgG. Lane 2 represents 12.5% of input soluble extract and (lane 3) represents 25% of input insoluble extract used for IP. Bars, 20 µm (A,D,G).

View larger version (92K): [in a new window]   Fig. 8. Ectopic Pax3 induces JNK activation and its accumulation in cytoplasmic multivesicular structures. Saos-2 cells were infected with control (Ad-ß-gal) (A,C) or Ad-Pax3flag (B,D) adenoviruses. At three days postinfection cells were fixed with paraformaldehyde (A,B and insets) or paraformaldehyde-cytoskeletal fixation (C,D), and the distribution of (activated) phospho-JNK was determined by indirect fluorescence microscopy. Arrowheads indicate the localization of activated JNK (A,C) and exogenously expressed HA-tagged JNK (C, inset) to focal adhesions in control infected cells. Ad-Pax3flag induces a large increase in activated JNK, which accumulates primarily in juxta-nuclear (B and arrow in D) or perinuclear (B, inset) multivesicular structures as well as at focal complexes (arrowheads in D). Ad-Pax3flag also induces juxtanuclear vesicular accumulation of exogenous HA-tagged JNK (arrow in D, inset). Images in C, D and respective insets are confocal images. Bars, 20 µm. (E) Ectopic Pax3 induces increased JNK expression. Total extracts of control or Ad-Pax3flag-infected Saos-2 cells were harvested postinfection at the indicated times and levels of total JNK proteins assessed by western blotting using an anti-JNK antibody. The same membrane was reprobed for actin as a loading control.

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