Law AL , Vehlow A , Kotini M , Dodgson L , Soong D , Theveneau E , Bodo C , Taylor E , Navarro C , Perera U , Michael M , Dunn GA , Bennett D , Mayor R , Krause M .

077429/Z/05/Z Wellcome Trust , 082907/Z/07/Z Wellcome Trust , 084659/Z/08/Z Wellcome Trust , BB/F011431/1 Biotechnology and Biological Sciences Research Council , BB/G00319X/1 Biotechnology and Biological Sciences Research Council , BB/J000590/1 Biotechnology and Biological Sciences Research Council , C20691/A11834 Cancer Research UK, C20691/A6678 Cancer Research UK, MR/J000655/1 Medical Research Council , RE/08/003 British Heart Foundation , G0401026 Medical Research Council

	Figure 1. Lamellipodin interacts with the Scar/WAVE complex. (A and B) Coimmunoprecipitation using Lpd or IgG control antibodies from HEK293 cell lysates expressing GFP-Lpd and the tagged Scar/WAVE complex including FLAG-WAVE1 (A) and Myc-WAVE2 (B). Myc-HSPC300 is not shown. (C) Endogenous Scar/WAVE1 and Lpd coimmunoprecipitate from lysates of primary cortical neurons using Lpd antibodies but not with IgG control. (D) Knockdown of Lpd by siRNA in B16F1 cells does not reduce expression of the Scar/WAVE complex (HSPC300 not shown) or Arp3. Loading control: Tubulin. (E–G) Endogenous Lpd (green) colocalizes with Scar/WAVE1 (E), Abi1 (F), and Sra1 (G; red) at the very edge of lamellipodia in B16F1 mouse melanoma cells. Representative line scan from multiple experimental repeats across the leading edge (location indicated on merged images) shows colocalization of Lpd (green) and Scar/WAVE1 (E), Abi1 (F), and Sra1 (G; red). Bar, 25 µm. See also Fig. S1.
	Figure 2. Lpd directly interacts with the SH3 domain of Abi. (A) Pull-down of Lpd from NIH/3T3 cell lysate using the GST-Abi-SH3 domain or GST as control. (B–D) Far Western overlay on different GST-Lpd truncation mutants (B) or GST control using purified (C) MBP-Abi1full-length or (D) MBP-Abi1ΔSH3 was detected with anti-MBP antibodies. Three independent experiments were performed. (E) Far-Western overlay with MBP-Abi1full-length on a peptide array covering the C terminus of Lpd with 12-mer peptides overlapping each other by three amino acids was detected with anti-MBP antibodies. (F) Table shows Abi SH3 domain–binding motifs in the Lpd sequence. The two GST-Lpd fragments highlighted in red correspond to the most strongly interacting Lpd fragments in the Far-Western experiment in C. The amino acid residues highlighted in yellow correspond to the core residues required for class II SH3 domain binding.
	Figure 3. The interaction between Lpd and the Scar/WAVE complex is mediated by the Abi SH3 domain and positively regulated by active Rac. (A–D) The Abi SH3 domain and three Abi binding sites in Lpd mediate the interaction between Lpd and the Scar/WAVE complex. Immunoprecipitation using Lpd antibodies or IgG control from HEK293 cell lysates (A) expressing GFP-Lpd and all Myc-tagged components of the Scar/WAVE complex, including Myc-Abi1full-length (A, left), Myc-Abi1ΔSH3 (A, right), or GFP-Abi and GFP-Lpd (C, left) or GFP-LpdAbiMut (C, right), show coimmunoprecipitation between Lpd and all components of the Scar/WAVE complex only when the Abi SH3 domain is present (A; Myc-HSPC300 is not shown) or between Lpd and GFP-Abi only when the Abi binding sites are present (B). Western blot: anti-GFP. (B and D) Comparison of efficiency of coimmunoprecipitation of Lpd with all components of the Scar/WAVE complex (B) or GFP-Lpd or GFP-LpdAbiMut with Abi (D). Quantification of band intensity of chemiluminescence imaged with a charge-coupled device camera. (B) Coimmunoprecipitation is reduced by >90%. Error bars indicate mean ± SEM, n = 3. One-way analysis of variance (ANOVA) and Tukey’s test were used; **, P < 0.01. (D) Coimmunoprecipitation is reduced by >94%. Error bars indicate mean ± SEM, n = 3. An unpaired t test was used; ***, P < 0.001. (E and F) Lpd and the RA-PH domains of Lpd are in complex with active Rac. Purified GTPγS- or GDPβS-loaded GST-Rac, or GST only as control, on Sepharose beads were incubated with lysates from HEK cells expressing GFP-Lpd (E) or GFP-Lpd-RAPH (F) and bound with GFP-Lpd or GFP-Lpd-RAPH. Samples were detected in a Western blot against GFP. (G) The RA-PH domains of Lpd directly interact with active Rac. Purified GTPγS- or GDPβS-loaded GST-Rac or GST only as control Sepharose beads were incubated with MBP-Lpd-RAPH or MBP only as control, and direct interaction was detected in a Western blot against MBP. (H and I) The interaction between Lpd and Abi is positively regulated by active Rac. Immunoprecipitation using Lpd-specific antibodies or IgG control from HEK293 cell lysates expressing GFP-Abi, GFP-Lpd, and dominant-active Rac (DA Rac; H, left) or dominant-negative Rac (DN Rac; H, middle), or empty vector control (H, right) show increased coimmunoprecipitation between Lpd and GFP-Abi only when dominant-active Rac is coexpressed. Western blot: anti-GFP. (I) Comparison of efficiency of coimmunoprecipitation of Lpd with GFP-Abi from blots in H. Quantification of band intensity of chemiluminescence imaged with a charge-coupled device camera. Coimmunoprecipitation is increased by >100% compared with empty vector and 150% compared with DN-Rac. Error bars indicate mean ± SEM, n = 3. One-way ANOVA and Tukey’s test were used. *, P < 0.05; **, P < 0.01.
	Figure 4. Lpd regulates cell spreading. (A and B) Western blot of cell lysates of Lpd WT and Lpd KO MEFs using anti-Lpd (A) or Scar/WAVE1 (B). Loading control: anti-HSC70. (C and D) F-actin staining (phalloidin) in Lpd WT (C) and Lpd KO MEFs (D). Arrows in C indicate the presence of lamellipodia in Lpd WT MEFs. Arrowheads in D indicate the absence of lamellipodia. (E and F) F-actin staining (phalloidin) determines the area of Lpd WT (E) and Lpd KO MEFs (F) after 60 min of spreading on fibronectin. (G) Quantification of the spreading area of MEFs from E and F. Values are mean ± SEM (error bars) of 131 (KO) or 155 (WT) cells. Unpaired, two-tailed t test: ****, P ≤ 0.0001. Bars, 25 µm.
	Figure 5. Lpd regulates cell migration via Abi and the Scar/WAVE complex. (A and B) Quantification of velocity (A) and persistence (B) of randomly migrating Lpd WT or KO MEFs. Mean population speed and persistence (dt = 2, TR = 4; see Materials and methods for calculation). Results are mean ± SEM (error bars), with three independent experiments. ****, P ≤ 0.0001, unpaired t test. (C and D) A confluent layer of WT or KO Lpd MEFs was scratched, and the area of the scratch measured at 0 and 24 h. Bar, 500 µm. Area closure is shown as the percentage of WT cells. (D) Results are mean ± SEM, with four independent experiments. ***, P ≤ 0.001, unpaired t test. See also Fig. S3 and Videos 1 and 2. (E and F) Lpd overexpression increases cell migration speed via Abi and Scar/WAVE. MDA-MB231 breast cancer cells, stably expressing Nap1-specific (Nap1 shRNA 1 or 2) or scrambled control shRNA were transiently transfected with GFP-Lpd or GFP as control (E) or GFP-Lpd, GFP-LpdEVMut, GFP-LpdAbiMut, GFP-LpdEV+AbiMut, or GFP as control (F). A confluent cell layer was scratched and the area of the scratch was measured at 0 and 24 h. Area closure is shown as percentage increase over GFP cells. Results are mean ± SEM (error bars), from three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant; one-way ANOVA was used. (E) Tukey’s test. (F) Newman-Keuls method.
	Figure 6. Lpd functions to regulate melanoblast migration. (A) Conditional Lpd KO mice were generated by flanking exon 4 with loxP sites. Cre-mediated recombination of the loxP sites results in the removal of exon 4, creating a frame shift between exon 3 and 5 and premature termination. (B and C) Conditional Lpd KO mice crossed with β-actin-Cre mice on a mixed genetic background produced mice with a reduced body size (−20.6 ± 3.0% SEM; ****, P ≤ 0.0001, unpaired t test), which also display missing pigmentation on the ventral side (D). (E and F) To visualize melanoblasts, DCT-LacZtg/tg;β-actin-Cretg/+;Lpdflox/flox whole-mount embryos at E14.5 were stained for β-galactosidase expression in the melanoblasts. (E) Areas within three 1 mm × 1.5 mm boxes positioned at the middle of the trunk between the fore and hind limbs were quantified in WT and KO animals. Bar, 1.5 mm. (F) Lpd KO mice show a significant reduction in the number of melanoblasts in all three boxes. Bar, 150 µm. Melanoblast numbers were reduced by ∼60%, ∼40%, and ∼30% for boxes 1, 2, and 3, respectively (20 KO or WT embryos from three litters; unpaired t test; *, P < 0.05; error bars indicate SEM). See also Fig. S4.
	Figure 7. Lpd regulates NC migration via Abi. (A) In situ hybridization for Twist (Hopwood et al., 1989; migratory NC marker) in control embryos or embryos injected with Lpd mRNA, Lpd, or Abi MOs (Lpd MO or Abi MO) or the dominant-negatives Lpd N1, Lpd N6, and Abi1ΔSH3. Black arrowheads, normally migrating NC streams. Red arrowheads, streams with impaired migration. Blue asterisks, the eye. (B) Summary of phenotypes. Lpd overexpression has no effect on overall NC cell migration. Lpd MO, Abi MO, and the dominant-negatives Lpd N1, Lpd N6, and Abi1ΔSH3 all impair NC cell migration. (C) Percentages of embryos with normal migration along the dorso-ventral axis (nctl = 26, nLpdRNA = 41, nLpdMO = 37, nLpdN1 = 26, nLpdN6 = 49, nAbiMO = 16, and nAbi1ΔSH3 = 24). (D) Mean distance of migration along the dorso-ventral axis as a percentage compared with control embryos (nctl = 12, nLpdRNA = 14, nLpdMO = 12, nLpdN1 = 14, nLpdN6 = 14, nAbiMO = 12, nAbi1ΔSH3 = 12). One-way ANOVA and Dunnett’s test were used. ***, P < 0.001. (E) In situ hybridization for Twist in Lpd or Abi (Lpd MO or Abi MO), Lpd mRNA and Lpd MO, or Abi mRNA and Abi MO or control MO injected embryos. (F) Mean distance of migration along the dorso-ventral axis compared with control MO embryos (nctl MO = 13, nLpdMO = 14, nLpdMO = 26, nLpdMO+LpdmRNA = 20, nAbiMO = 19, nAbiMO+AbimRNA = 26). One-way ANOVA and Dunnett’s test were used. **, P < 0.01; ns, nonsignificant. (G–I) Lpd and Abi function cell-autonomously in NC migration. (G) Schematic diagram of graft experiment in H. (H) NC from MO-treated embryos or WT embryos was grafted into WT or MO-treated embryos, and NC migration was analyzed. (I) Quantification of NC migration phenotypes from H. Numbers (embryos with inhibition of NC migration/total): nctl>ctl = 0/4 (0%), nLpdMO>ctl = 4/6 (66%), nctl>LpdMO = ¼ (25%), nAbiMO>ctl = 4/5 (80%), nctl>AbiMO = 0/5 (0%). Bars, 150 µm.
	Figure 8. Lpd regulates lamellipodia protrusion and cell migration via Abi in Xenopus NC cells. (A) Localization of Lpd-GFP in Xenopus NC cells cultured on fibronectin. Actin filaments are stained with TRITC-Phalloidin. (B) Cells expressing nuclear mCherry (pseudocolored magenta by thresholding) and membrane GFP (pseudocolored green by thresholding) were used to analyse cell protrusions (pseudocolored red). Cell protrusions were defined as the area of protrusion that extends beyond the cell body. Cell protrusions are shown for control cells and cells injected with Lpd mRNA, Lpd N6 or co-injected with Lpd mRNA and Abi-Δ-SH3. Note that Lpd overexpression leads to enlarged protrusions. This effect is abolished by co-injection with Abi-Δ-SH3.. (C–E) Area of cell protrusions expressed as a proportion of normal protrusions (graph in D: nctl = 58, nLpd800pg = 33, nAbi1ΔSH3 = 24, nLpd+Abi1ΔSH3 = 17 [one-way ANOVA and Dunnett’s test; ***, P < 0.001]; graph in E: nctl = 154, nLpd400pg = 148, nLpd800pg = 157, nLpdN6 = 121 [one-way ANOVA and Tukey’s test; ***, P < 0.001]). (F–J) Tracks of NC cells cultured on fibronectin (a subset of the analyzed tracks are shown). (K and L) Velocity and persistence from tracks performed on all conditions (nctl = 543, nLpdMO = 240, nLpdmRNA = 297, nAbiMO = 180, nLpd+AbiMO = 290). One-way ANOVA and Dunnett’s test were used. *, P < 0.05; ***, P < 0.001. Error bars indicate SEM. See also Fig. S5 and Videos 3–6.
	Figure 9. pico regulates Drosophila border cell migration via SCAR. (A) GST-Pico, the fly orthologue of Lpd, pulled down all Myc-tagged components of the Scar/WAVE complex. Myc-HSPC300 is not shown. (B) pico knockdown or overexpression, or SCAR RNAi under the control of slbo-GAL4 abrogate migration at stage 9. (B, top) Representative images of WT and defective egg chambers. Green, GFP-labeled border cells; red, DNA; blue, F-actin. The box and whiskers plot shows mean border cell position: distance of the cluster relative to the most anterior position of the overlying follicle cells (broken lines). Top and bottom box: 75th and 25th quartile; whiskers indicate minimum and maximum. One-way ANOVA and Dunnett’s test were used. ***, P < 0.001; n = 50. (C) pico overexpression abrogates migration at stage 10A, and this is ameliorated by SCAR RNAi. Histogram summarizes migration defects in the indicated genotypes. Migration was calculated as a percentage of the distance traveled to the oocyte/nurse cell boundary (broken lines in top panels). For each genotype, n = 50 egg chambers.
	Figure 10. pico regulates Drosophila border cell migration via SCAR. (A–D) Analysis of time-lapse images of LifeAct-GFP–labeled border cells, using c306-GAL4 to drive the indicated genotypes (graphs indicate mean ± SEM; WT, n = 9; PVRDN, n = 9; pico RNAi, n = 12; pico OE, n = 12; SCAR RNAi, n = 8; SCAR RNAi, pico OE, n = 7). (A) Graph summarizing migration rate/frame calculated using a custom macro (Poukkula et al., 2011). (B) Graph showing percentage frames from the first half of migration with tumbling border cell clusters (see Materials and methods). (B, right) GFP-labeled clusters display a polarized or tumbling phenotype. Bar, 15 µm. (A and B) Tests used were one-way ANOVA (P < 0.0001) and Tukey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Graph showing number of cellular extensions per frame, irrespective of their direction. One-way ANOVA and Dunnett’s test were used. *, P < 0.05; **, P < 0.01. (D) Graph summarizing percentage extensions/frame at front, back, or sides of the cluster. (D, right) Image of a border cell cluster before and after image segmentation. The body of the cluster is shown in blue, cellular extensions at the front in green, the back in red, and the sides in white. White lines indicate quadrants, representing front, back, and sides for quantification. Bar, 5 µm.

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