XB-ART-47766J Cell Biol November 25, 2013; 203 (4): 673-89.
Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo.
Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd''s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.
PubMed ID: 24247431
PMC ID: PMC3840943
Article link: J Cell Biol
Genes referenced: abi1 actl6a actr3 cyfip1 dct fn1 gnl3 hspa8 lgals4.2 mbp myc npat rac1 scaf1 sox9 sra1 tab3 tbx2 twist1 wasf1 wasf2
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|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 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.|