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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

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 of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl2, 0.5 mM EGTA. (B) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). (C) SDS-PAGE as in Figure 1H, showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). (D) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl2, and 0.5 mM EGTA. (C, D) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification.DOI:http://dx.doi.org/10.7554/eLife.06585.004
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fig1s1: 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 of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl2, 0.5 mM EGTA. (B) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). (C) SDS-PAGE as in Figure 1H, showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). (D) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl2, and 0.5 mM EGTA. (C, D) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification.DOI:http://dx.doi.org/10.7554/eLife.06585.004

Mentions: Lpd takes its name from the dynamic lamellipodial actin networks to which it localizes in vivo, even in the absence of Ena/VASP proteins or free actin filament barbed ends (Krause et al., 2004). This tenacious localization to leading edge actin networks suggested that Lpd might interact directly with actin filaments, so we tested this idea by simultaneously visualizing monomeric GFP-Lpd850−1250aa and individual actin filaments in vitro by Total Internal Reflection Fluorescence (TIRF) microscopy (Figure 1A,B). In buffer containing 50 mM KCl, the GFP-labeled, monomeric Lpd construct uniformly decorated actin filaments, with a measured dissociation equilibrium constant (Kd) of 255 ± 2 nM (Figure 1B,C). Consistent with a weak electrostatic interaction, Lpd binding to filamentous actin grew progressively weaker in buffers containing higher concentrations of KCl. In the presence of 100 mM KCl, interaction between monomeric Lpd and single actin filaments were undetectable by TIRF-M (Figure 1B). In contrast to these single-filament TIRF assays, we were able to detect interactions between Lpd850−1250aa and filamentous actin by co-sedimentation in the presence of physiological salt concentrations (Figure 1—figure supplement 1A,B). The stronger actin filament binding observed by co-sedimentation likely results from Lpd bundling actin filaments in solution.10.7554/eLife.06585.003Figure 1.Lamellipodin (Lpd) binds directly to single actin filaments in vitro.


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

Hansen SD, Mullins RD - Elife (2015)

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 of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl2, 0.5 mM EGTA. (B) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). (C) SDS-PAGE as in Figure 1H, showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). (D) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl2, and 0.5 mM EGTA. (C, D) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification.DOI:http://dx.doi.org/10.7554/eLife.06585.004
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Related In: Results  -  Collection

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fig1s1: 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 of 2 µM filamentous actin (+4 µM dark phalloidin) in the presence of 1, 2, and 4 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (monomeric protein concentration). Buffer composition is 20 mM HEPES [pH 7], 50–150 mM KCl, 0.5 mM ATP, 0.5 mM MgCl2, 0.5 mM EGTA. (B) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50, 100, and 150 mM KCl. Error bars represent S.D. of the mean (n = 3 experiments). (C) SDS-PAGE as in Figure 1H, showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd (0–10 µM monomer concentration). (D) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments. Actin was polymerized at a concentration of 20 µM in the absence (termed ‘native’) or presence of an equal molar concentration of dark phalloidin (indicated by ‘+’). After 45 min, filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa and incubated for 1 hr before ultracentrifugation (also see ‘Materials and methods’). The final buffer composition was 20 mM HEPES [pH 7.0], 100 mM KCl, 1 mM TCEP, 0.5 mM ATP, 0.5 mM MgCl2, and 0.5 mM EGTA. (C, D) Asterisks (*) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification.DOI:http://dx.doi.org/10.7554/eLife.06585.004
Mentions: Lpd takes its name from the dynamic lamellipodial actin networks to which it localizes in vivo, even in the absence of Ena/VASP proteins or free actin filament barbed ends (Krause et al., 2004). This tenacious localization to leading edge actin networks suggested that Lpd might interact directly with actin filaments, so we tested this idea by simultaneously visualizing monomeric GFP-Lpd850−1250aa and individual actin filaments in vitro by Total Internal Reflection Fluorescence (TIRF) microscopy (Figure 1A,B). In buffer containing 50 mM KCl, the GFP-labeled, monomeric Lpd construct uniformly decorated actin filaments, with a measured dissociation equilibrium constant (Kd) of 255 ± 2 nM (Figure 1B,C). Consistent with a weak electrostatic interaction, Lpd binding to filamentous actin grew progressively weaker in buffers containing higher concentrations of KCl. In the presence of 100 mM KCl, interaction between monomeric Lpd and single actin filaments were undetectable by TIRF-M (Figure 1B). In contrast to these single-filament TIRF assays, we were able to detect interactions between Lpd850−1250aa and filamentous actin by co-sedimentation in the presence of physiological salt concentrations (Figure 1—figure supplement 1A,B). The stronger actin filament binding observed by co-sedimentation likely results from Lpd bundling actin filaments in solution.10.7554/eLife.06585.003Figure 1.Lamellipodin (Lpd) binds directly to single actin filaments in vitro.

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