(A) Cartoon representation of the human Lpd850−1250aa highlighting the Enabled/Vasodilator (Ena/VASP) binding sites (grey), Abi1/endophilin SH3 binding sites (red), and basic amino acid residues …
(A) Monomeric GFP-Lpd850−1250aa and dimeric GFP-LZ-Lpd850−1250aa interact with filamentous actin in the presence of 50, 100, 150 mM KCl. SDS-PAGE from three experiments showing the cosedimentation …
(A) Protein sequence alignment of human Lpd and homologs C-termini. Basic amino acid residues (arginine and lysine) are highlighted in blue. Gray boxes mark the location of the canonical Ena/VASP …
(A) Cartoon illustrating his10Cherry-SCARAPWCA, his10GFP-Lpd850−1250aa, and his10GFP tethered to a lipid coated beads (LCBs) containing DOGS-NTA(Ni) lipid (blue head groups). Actin network assembly …
(A) Montage of actin comet tails frozen at different times point with 37.5 µM Latrunculin B-phalloidin containing buffer. Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), …
(A, B) Plasma membrane localization of GFP-Lpd1−1250aa visualized with TIRF microscopy in Xenopus Tissue Culture (XTC) cells spread on poly-L-lysine (PLL). GFP-Lpd1−1250aa was ectopically expressed …
(A, C) Representative images showing the plasma membrane localization of (A) GFP-Lpd1−1250aa, (B) GFP-Lpd850−1250aa, and (C) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells …
(A, B) Representative kymographs showing retrograde flow of (A) monomeric GFP-Lpd850−1250aa and (B) mCherry-Actin in XTC cells. Images were acquired every 5 s. Scale bars, 5 µm and 5 min. (C, D) …
(A) Lpd FPPPP peptide sequences are required to recruit Ena/VASP proteins to the lipid coated beads (LCBs). Glass microspheres were coated with SUVs containing DOPC/DOGS Ni-NTA lipids (96:4 molar …
(A) Cartoon showing domain organization of human Lpd (1–1250aa). (B) Analytical ultracentrifugation sedimentation equilibrium traces for GFP-Lpd850−1250aa in the absence (left) and presence of 10, …
Protein sequence alignment of Lpd850−1250aa wild-type and mutants highlighting the separation of function mutations targeting either the actin BD (arg/lys; BLUE), Ena/VASP binding sites (GRAY), or …
(A) Plasma membrane localization of Lyn-GFP-Lpd850−1250aa, wild-type and mutants, visualized using TIRF microscopy. Mutations affecting the interaction with either Ena/VASP proteins (AAPPP)x6 or …
(A) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization. Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell …
(A) VASP EVH1 and FL VASP mutants cannot interact with actin filaments in vitro. Images highlight the inability of 200 nM (monomeric concentration) wild-type Cy3-VASP1−380aa, Cy3-VASP1−114aa (EVH1 …
(A) Montage of single actin filaments polymerizing in the presence of 2 µM actin (20% Cy5 labeled) and TIRF buffer containing 100 mM KCl. Compared to actin filaments elongating in the presence of 50 …
(A) Monomeric actin antagonizes GFP-Lpd850−1250aa actin filament binding. Visualization of 500 nM GFP-Lpd850−1250aa in the absence or presence of 4 µM monomeric actin in the presence of buffer …
(A, B) Lpd bundles dynamically elongating actin filaments. Montage showing single actin filaments elongating and bundling in the presence of 2 µM actin (5% Cy5 labeled) and either (A) 1 µM GFP-Lpd850…
Based on the canonical model (Krause et al., 2004), Lpd is recruited to actin based membrane protrusions through interactions with phosphatidylinositol lipids (i.e., PI(3,4)P2) and possibly small …
Images were acquired with temporal resolution of 100 ms using Total Internal Reflection Fluorescence (TIRF)-M. Video plays at 15 frames per second. Scale bar, 5 µm.
Cell was imaged with a temporal resolution of 5 s using TIRF-M at 23°C. GFP-Lpd850−1250aa and mCherry-Actin were ectopically expressed from either a CMV or DeltaCMV promoter, respectively. Video …
B16F1 cell was imaged with a temporal resolution of 10 s using wide-field epi fluorescence at 37°C. GFP-Lpd850−1250aa was ectopically expressed from a CMV promoter. Video plays at 10 frames per …
Images were acquired using TIRF microscopy with temporal resolution of 50 ms time. Video plays at 15 frames per second. Scale bar, 5 µm.
Images were acquired with a temporal resolution of 10 s using TIRF-M at 23°C. Drugs were added after 150 s of imaging. Video plays at 10 frames per second. Scale bar, 5 µm.
Cytochalasin D was added at 214 s. Video plays at 50 frames per second. Scale bar, 5 µm.
Cytochalasin D was added at 102 s. Video plays at 50 frames per second. Scale bar, 5 µm.
For clarity, only GFP-LZ-Lpd (850−1250aa) is shown. Images were acquired with temporal resolution of 0.5 s using TIRF-M. Video plays at 50 frames per second. Scale bar, 5 µm.
Elongation of single actin filaments were visualized in the presence of 2 µM Mg-ATP actin (20% Cy5 labeled), 5 nM Cy3-VASP, 50 nM his10-GFP-Lpd850−1250aa, and buffer containing 75 mM KCl. Images …
Table of plasmid DNA used for protein expression and cellular transfections.