Scale bars, 5 µm and 1 min. DOI: http://dx.doi.org/10.7554/eLife.06585.010 To determine which Lpd binding partners actin, Ena/VASP, Abi1/endophilin, inositol phospholipids, or Ras/Rho-family G-proteins are required for leadinG-proteins areation, we comparequiredlocalization oforarious leadingncation mutants in XTC cedge.
To determine the role of membrane association in the leading-edge localization of Lpd we attempted to rescue leading edge localization of various GFP-Lpd850−1250aa mutants by constitutively tethering each protein to the plasma membrane with a Lyn palmitolyation sequence derived from Src kinase (Inoue et al., 2005).
In each case, mutations in the Ena/VASP or Abi1/endophilin binding sites resulted in a loss of leading edge localization that coincided with an increased cytoplasmic localization of the GFP-Lpd850−1250aa construct.
However, one distinction from our in vitro results is that the R28D mutation was sufficient to block leading-edge localization in vivo, whereas the 2×KE mutant Coro1C still retained leading edge localization.
At some stage the amplitude of the wrinkles starts to grow exponentially with the number of cycles N leading to localization and collapse.
Different hypotheses have been proposed to explain this behavior, but there is still no straightforward relation between the particular stress and strain state induced by SPIF and the material degradation leading to localization and fracture.
Live movies in another cell type, such as B16F1 should be performed to confirm the findings for leading edge localization.
DOI: http://dx.doi.org/10.7554/eLife.06585.016 10.7554/eLiFigure85 figuregure 5—figure supplement 3. Membrane tethered Lyn-GFP-Lpd (850 1250aa) requires basic residue for leading edge localization.
Mutating all lysine and arginine residues (44A) or only those outside of the Abi1/Endophilin SH3 BDs (35A) eliminated the leading edge localization of Lyn- GFP-Lpd850−1250aa.
(B ) Basic residues in flanking the Ena/VASP and Abi1/endophilin binding sites are required for leading edge localization of GFP-Lpd850−1250aa in XTC cells.
