Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments
- University of California, San Francisco School of Medicine, United States
- Howard Hughes Medical Institute, University of California, United States
Figures
Figure 1 with 1 supplement
Lamellipodin (Lpd) binds directly to single actin filaments in vitro.
(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 …
Figure 1—figure supplement 1
Interactions between filamentous actin, GFP-Lpd (850–1250aa), and GFP-LZ-Lpd (850–1250aa) measured by cosedimentation at different buffer ionic strengths.
(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 …
Figure 2
Conservation of Lpd (850–1250aa) amino acid sequence and isoelectric point (pI).
(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 …
Figure 3 with 1 supplement
Membrane-tethered Lpd slows dendritic actin network assembly in vitro.
(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 …
Figure 3—figure supplement 1
Actin based motility on lipid coated glass beads.
(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), …
Figure 4 with 2 supplements
Lpd (850–1250aa) localizes to the leading edge membranes and undergoes retrograde flow with the actin cytoskeleton.
(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 …
Figure 4—figure supplement 1
Localization of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa).
(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 …
Figure 4—figure supplement 2
Retrograde flow of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa) with the actin cytoskeleton.
(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) …
Figure 5 with 3 supplements
Interactions with Ena/VASP or Abi1/endophilin are not required for Lpd (850–1250aa) membrane localization.
(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 …
Figure 5—figure supplement 1
Lpd-VASP binding stoichiometry determined by sedimentation equilibrium.
(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, …
Figure 5—figure supplement 2
Lpd (850–1250aa) wild-type and mutant protein sequence alignment.
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 …
Figure 5—figure supplement 3
Membrane tethered Lyn-GFP-Lpd (850–1250aa) requires basic residue for leading edge localization.
(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 …
Figure 6
Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).
(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 …
Figure 7 with 1 supplement
Lpd can simultaneously interact with VASP and filamentous actin.
(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 …
Figure 7—figure supplement 1
Lpd and VASP synergistically bundle actin filaments.
(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 …
Figure 8 with 1 supplement
Lpd enhances VASP barbed end processivity.
(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 …
Figure 8—figure supplement 1
Lpd dependent actin filament bundling.
(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…
Figure 9
Model.
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 …
Videos
Video 1
Localization of full length GFP-Lpd (1 −1250aa) expressed at single molecule concentrations from the DeltaCMV promoter in Xenopus Tissue Culture (XTC) cells.
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.
Video 2
Retrograde flow of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells spread on poly-L-lysine (PLL) coated coverslips.
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 …
Video 3
Leading edge membrane localization of GFP-Lpd850−1250aa in polarized B16F1 mouse melanoma cell migrating on laminin coated glass.
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 …
Video 4
Localization of membrane tethered Lyn-GFP-Lpd (850−1250aa) at the leading edge membrane of XTC cells spread on PLL coated coverslips expressed at single molecule concentrations from DeltaCMV promoter.
Images were acquired using TIRF microscopy with temporal resolution of 50 ms time. Video plays at 15 frames per second. Scale bar, 5 µm.
Video 5
Localization of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B.
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.
Video 6
Localization of GFP-Lpd850−1250aa following the addition of 100 nM Cytochalasin D in a XTC cell imaged with a temporal resolution of 2 s using TIRF-M at 23°C.
Cytochalasin D was added at 214 s. Video plays at 50 frames per second. Scale bar, 5 µm.
Video 7
Localization of GFP-LZ-Lpd850−1250aa following the addition of 100 nM Cytochalasin D in a XTC cell imaged with a temporal resolution of 2 s using TIRF-M at 23°C.
Cytochalasin D was added at 102 s. Video plays at 50 frames per second. Scale bar, 5 µm.
Video 8
Dynamic actin filament association of 200 nM dimeric GFP-LZ-Lpd (850−1250aa) in the presence of 2 µM Mg-ATP (20% Cy5 labeled) visualized by TIRF-M.
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.
Video 9
Visualization of a processive VASP-Lpd tip complex bound to the barbed end of a single actin filament.
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 …
Additional files
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Supplementary file 1
Table of plasmid DNA used for protein expression and cellular transfections.
- https://doi.org/10.7554/eLife.06585.030
