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Files in this Data Supplement:
Fig. S1. ABBA is highly conserved protein in vertebrates and possesses an N-terminal IM domain. (A) Amino acid alignment of mouse IM domains. Accession numbers: IRSp53 ENSMUSP00000026436; IRTKS ENSMUSP00000018270; ABBA ENSMUSP00000050211; MIM ENSMUSP00000079239; FLJ22582 ENSMUSP00000053129. (B) Sequence alignment of mouse (Mus Musculus), human (Homo sapiens), fish (three-spined stickleback, Gasterosteus aculeatus) and frog (Xenopus tropicalis) ABBA proteins. The serine-rich region is underlined with a green bar. The proline-rich motifs are boxed with a red line, the leucine-zipper motif is indicated with black dots, and the sequence corresponding to the WH2 domain is underlined with an orange bar. Black box indicates the region responsible for the auto-regulatory functions of the IM domain. The mutations generated in this work are indicated with red arrowheads and highlighted with ellipses. The coloring and (*), (:), (.) symbols reflect the degree of conservation between the sequences. Accession numbers: Mus musculus ENSMUSP00000050211; Homo sapiens ENSP00000341171; Xenopus tropicalis ENSXETP00000034591; Gasterosteus aculeatus ENSGACP00000018828. Sequence alignments were made using ClustalX.
Fig. S2. Polyclonal ABBA antibody does not crossreact with MIM or IRSp53. NIH3T3 cells were transfected with constructs expressing GFP-fusions of ABBA, MIM and IRSp53. Equal amounts of cell lysates were loaded on SDS-PAGE gel, blotted and stained with polyclonal ABBA antibody. The antibody recognized only GFP-ABBA but not GFP-MIM or GFP-IRSp53. Ponceau red and anti-actin staining is shown as a loading control.
Fig. S3. ABBA interacts with active and inactive Rac trough its IM domain. (A) Lysates of HeLa cells overexpressing GFP-tagged ABBA were incubated with recombinant GTPases coupled to glutathione-Sepharose beads followed by SDS-PAGE and immunoblotting with ABBA antibody. (B) Recombinant ABBA-IMD was incubated with recombinant GTPases coupled to glutathione-Sepharose beads at physiological ionic conditions. The beads were washed three times followed by SDS-PAGE analysis.
Fig. S4. Localization of ABBA to the cortical plasma membrane is dependent on the lipid-binding interface. (A-C) GFP-tagged full-length ABBA localizes to the peripheral plasma membrane (white arrowhead) and to the dorsal membrane ruffles (black arrowhead) similarly to the endogenous protein. (D-F) Mutation in the IM domain abolishes the localization of ABBA in the cell periphery but not from dorsal ruffles. (G-I) Mutation in the actin-binding surface of the WH2 domain does not disturb the localization of ABBA. (J-L) Deleting the two central proline-rich regions (508-587) does not have an effect in the localization of ABBA. White box shows magnifications of the indicated region of the image. Scale bar: 20 µm.
Fig. S5. Disruption of the actin cytoskeleton by latrunculin A treatment inhibits ABBA-IMD induced filopodia extension. Quantification of the number of extending filopodia from three untreated C6-R cells and from the same cells 5 minutes after addition of 0.2 µg/ml latrunculin A, a drug that prevents actin polymerization. Extension of filopodia was observed over a 60 second time period. Data are represented as mean values ± s.e.m.
Fig. S6. ABBA-IMD does not localize in F-actin bundles. GFP-ABBA IM-domain (green) does not localize to peripheral F-actin (red) bundles as indicated by the arrowhead. In filopodia, ABBA surrounds the F-actin bundle but no colocalization (in yellow) is seen (arrow). Scale bar 2 µm.
Movie 1. GFP-tagged ABBA localizes to the plasma membrane in C6-R cells but does not induce microspikes. Speed is 15 frames/second. Frames were captured every 20 seconds.
Movie 2. GFP-tagged ABBA IM-domain induces massive microspike formation when expressed in cells. Speed is 15 frames/second. Frames were captured every 20 seconds.
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