Limits...
Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

Hansen SD, Mullins RD - Elife (2015)

Bottom Line: We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity.We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments.This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco School of Medicine, San Francisco, United States.

ABSTRACT
Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

No MeSH data available.


Related in: MedlinePlus

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, 25, 50, 75, 100 µM VASP1−114aa EVH1 domain. (C) Table showing the predicted and observed molecular weight of GFP-Lpd850−1250aa in absence and presence of different VASP1−114aa EVH1 domain protein concentrations. (D) Cartoon showing GFP-Lpd850−1250aa with VASP1−114aa EVH1 domains (grey spheres) binding to FPPPP sites (red triangles). Based on the observed binding stoichiometry between GFP-Lpd850−1250aa and VASP1−114aa, we hypothesize that steric hindrance allows only a single EVH1 domain to interact with the tandem FPPPP motifs (i.e., SPDFPPPPPESSLVFPPPPPSPVPA and SVVEFPSPPSDSDFPPPPPETD). The crystal structure of GFP was derived from Yang et al. (1996) (1GFL.pdb).DOI:http://dx.doi.org/10.7554/eLife.06585.015
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543927&req=5

fig5s1: 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, 25, 50, 75, 100 µM VASP1−114aa EVH1 domain. (C) Table showing the predicted and observed molecular weight of GFP-Lpd850−1250aa in absence and presence of different VASP1−114aa EVH1 domain protein concentrations. (D) Cartoon showing GFP-Lpd850−1250aa with VASP1−114aa EVH1 domains (grey spheres) binding to FPPPP sites (red triangles). Based on the observed binding stoichiometry between GFP-Lpd850−1250aa and VASP1−114aa, we hypothesize that steric hindrance allows only a single EVH1 domain to interact with the tandem FPPPP motifs (i.e., SPDFPPPPPESSLVFPPPPPSPVPA and SVVEFPSPPSDSDFPPPPPETD). The crystal structure of GFP was derived from Yang et al. (1996) (1GFL.pdb).DOI:http://dx.doi.org/10.7554/eLife.06585.015

Mentions: We next asked whether GFP-Lpd850−1250aa requires Ena/VASP binding for leading edge membrane localization in XTC cells. The C-terminal region of Lpd contains six FPPPP peptide sequences, each of which can potentially bind one VASP EVH1 domain. By analytical ultracentrifugation, we found that GFP-Lpd850−1250aa simultaneously binds up to four VASP EVH1 domains in solution (Figure 5—figure supplement 1). We mutated all six FPPPP motifs in Lpd850−1250aa to AAPPP (Figure 5—figure supplement 2) and expressed this mutant protein as either a his10-GFP fusion in Escherichia coli (his10-GFP-Lpd(AAPPPx6)850−1250aa) or a GFP fusion in XTC cells (GFP-Lpd(AAPPPx6)850−1250aa). Although the his10-GFP-Lpd(AAPPPx6)850−1250aa mutant failed to recruit Ena/VASP proteins to LCBs in vitro (Figure 5A), the localization of the GFP-Lpd(AAPPPx6)850−1250aa mutant in XTC cells was nearly indistinguishable from GFP-Lpd850−1250aa (Figure 5B). The only noticeable difference was a slight decrease in the amount of protein associated with nascent focal adhesions (Figure 5B).10.7554/eLife.06585.014Figure 5.Interactions with Ena/VASP or Abi1/endophilin are not required for Lpd (850–1250aa) membrane localization.


Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments.

Hansen SD, Mullins RD - Elife (2015)

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, 25, 50, 75, 100 µM VASP1−114aa EVH1 domain. (C) Table showing the predicted and observed molecular weight of GFP-Lpd850−1250aa in absence and presence of different VASP1−114aa EVH1 domain protein concentrations. (D) Cartoon showing GFP-Lpd850−1250aa with VASP1−114aa EVH1 domains (grey spheres) binding to FPPPP sites (red triangles). Based on the observed binding stoichiometry between GFP-Lpd850−1250aa and VASP1−114aa, we hypothesize that steric hindrance allows only a single EVH1 domain to interact with the tandem FPPPP motifs (i.e., SPDFPPPPPESSLVFPPPPPSPVPA and SVVEFPSPPSDSDFPPPPPETD). The crystal structure of GFP was derived from Yang et al. (1996) (1GFL.pdb).DOI:http://dx.doi.org/10.7554/eLife.06585.015
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543927&req=5

fig5s1: 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, 25, 50, 75, 100 µM VASP1−114aa EVH1 domain. (C) Table showing the predicted and observed molecular weight of GFP-Lpd850−1250aa in absence and presence of different VASP1−114aa EVH1 domain protein concentrations. (D) Cartoon showing GFP-Lpd850−1250aa with VASP1−114aa EVH1 domains (grey spheres) binding to FPPPP sites (red triangles). Based on the observed binding stoichiometry between GFP-Lpd850−1250aa and VASP1−114aa, we hypothesize that steric hindrance allows only a single EVH1 domain to interact with the tandem FPPPP motifs (i.e., SPDFPPPPPESSLVFPPPPPSPVPA and SVVEFPSPPSDSDFPPPPPETD). The crystal structure of GFP was derived from Yang et al. (1996) (1GFL.pdb).DOI:http://dx.doi.org/10.7554/eLife.06585.015
Mentions: We next asked whether GFP-Lpd850−1250aa requires Ena/VASP binding for leading edge membrane localization in XTC cells. The C-terminal region of Lpd contains six FPPPP peptide sequences, each of which can potentially bind one VASP EVH1 domain. By analytical ultracentrifugation, we found that GFP-Lpd850−1250aa simultaneously binds up to four VASP EVH1 domains in solution (Figure 5—figure supplement 1). We mutated all six FPPPP motifs in Lpd850−1250aa to AAPPP (Figure 5—figure supplement 2) and expressed this mutant protein as either a his10-GFP fusion in Escherichia coli (his10-GFP-Lpd(AAPPPx6)850−1250aa) or a GFP fusion in XTC cells (GFP-Lpd(AAPPPx6)850−1250aa). Although the his10-GFP-Lpd(AAPPPx6)850−1250aa mutant failed to recruit Ena/VASP proteins to LCBs in vitro (Figure 5A), the localization of the GFP-Lpd(AAPPPx6)850−1250aa mutant in XTC cells was nearly indistinguishable from GFP-Lpd850−1250aa (Figure 5B). The only noticeable difference was a slight decrease in the amount of protein associated with nascent focal adhesions (Figure 5B).10.7554/eLife.06585.014Figure 5.Interactions with Ena/VASP or Abi1/endophilin are not required for Lpd (850–1250aa) membrane localization.

Bottom Line: We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity.We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments.This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco School of Medicine, San Francisco, United States.

ABSTRACT
Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

No MeSH data available.


Related in: MedlinePlus