JCS03384 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Immunostaining of additional Arp2/3 subunits in growth cones. Bar, 10 μm. (A) DRG growth cones immunostained against the p34 subunit and phalloidin. The staining pattern is the same as that observed for Arp3. (B,C) PC-12 growth cones immunostained against Arp3 (B) or p34 (C) and phalloidin. The staining pattern is the same as that observed in chick DRG growth cones.

• Supplemental Figure 2 -

  Fig. S2. Immunostaining controls. (A) Immunoblot of PC-12 lysate, probed with anti-p34 and tubulin (left), anti-Arp3 and tubulin (right) primary antibodies. (B) PC-12 cell extracted, fixed, and immunostained with rabbit non-immune serum in place of primary antibody and FITC secondary antibody (left), and rhodamine phalloidin (right) shows little fluorescence in the non-immune channel, with none observed in the growth cones. Bar, 10 μm. (C) Linescan analysis of both channels in B shows fluorescence in non-immune channel is negligible. (D) (left) NG-108 cells. The lower cell (*) is expressing myc-tagged Arp3, the white box marks a region containing the lamellipodia of both cells shown at higher magnification in the three panels to the right. The left center panel is immunostained against Arp3, and shows smooth staining at the leading edge of both cells. The right center panel is immunostained against myc, showing staining at the leading edge of the transfected cell, but not the untransfected cell. The merge in the right panel shows colocalization of Arp3 and myc immunostaining at the leading edge in the transfected cell. Bar, 5 μm. (E) Control immunogold EM, processed along with experimental samples, incubated with gold labeled secondary antibodies only. Grid marks 0.2 μm² boxes; number of gold particles contained in each box is shown.

• Supplemental Figure 3 -

  Fig. S3. Localization of GFP-Arp3 expressed in PC-12 cells using TIRF microscopy. (A) (left) Whole PC-12 cell expressing GFP-Arp3 imaged using widefield fluorescence microscopy, the central region of the cell body is grossly overexposed in order to visualize the three peripheral growth cones, giving the impression of enrichment in the central region. (right) The same cell imaged using TIRF microscopy a few seconds later, yielding an even optical field that reveals no enrichment in the central region. Bar, 5 μm. (B) For all images, red arrows indicate protrusion, cyan arrows indicate retraction. (a) Arp3 is seen in veils that protrude from the crotch of two pre-existing filopodia, away from the growth cone. (b) Arp3 is seen in veil protrusion along a single filopodium. (c) GFP-Arp3 is enriched in veils at the leading edge of the growth cone. (d) Arp3 enrichment decreases in retracting veils. Bar, 2 μm.

• Supplemental Figure 4 -

  Fig. S4. Localization of Arp3-immunogold particles to ‘Y’ junctions of protruding DRG veils. Bar, 0.1 μm.