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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

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-Lpd850−1250aa or (B) 1 µM GFP-LZ-Lpd850−1250aa (dimer concentration equals 0.5 µM) in TIRF buffer containing 50 mM KCl. Note that the intensity of single actin filaments is faint due to the low percentage of Cy5-labeled actin and low laser intensity used to prevent camera pixel saturation by the bright actin filament bundles. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.026
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fig8s1: 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-Lpd850−1250aa or (B) 1 µM GFP-LZ-Lpd850−1250aa (dimer concentration equals 0.5 µM) in TIRF buffer containing 50 mM KCl. Note that the intensity of single actin filaments is faint due to the low percentage of Cy5-labeled actin and low laser intensity used to prevent camera pixel saturation by the bright actin filament bundles. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.026

Mentions: Because human VASP is a weakly processive actin polymerase and Lpd can interact simultaneously with both VASP tetramers and actin filaments, we hypothesized that Lpd might modulate the processivity of VASP by both clustering tetramers and tethering them to actin filaments. We tested this hypothesis by visualizing actin filament elongation in the presence of either monomeric (Lpd850−1250aa) or dimeric (LZ-Lpd850−1250aa) GFP-Lpd constructs. The weak interaction between monomeric GFP-Lpd850−1250aa and filamentous actin turns out to be further antagonized by the presence of monomeric actin or profilin-actin (Figure 8A). Also, we observed a reduction in the rate of barbed end actin filament elongation in the presence 2 µM profilin-actin and soluble GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (Figure 8B), consistent with Lpd binding to (and partially sequestering) actin monomers. In contrast to monomeric Lpd, dimeric GFP-LZ-Lpd850−1250aa could transiently interact with both the sides and barbed ends of single growing actin filaments (Figure 8D, Video 8). Although GFP-Lpd and GFP-LZ-Lpd associate more transiently with single actin filaments in the presence of monomeric actin, Lpd binds well enough to promote filament bundling when present at a near stoichiometric concentration relative to monomeric actin (Figure 8—figure supplement 1).10.7554/eLife.06585.025Figure 8.Lpd enhances VASP barbed end processivity.


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

Hansen SD, Mullins RD - Elife (2015)

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-Lpd850−1250aa or (B) 1 µM GFP-LZ-Lpd850−1250aa (dimer concentration equals 0.5 µM) in TIRF buffer containing 50 mM KCl. Note that the intensity of single actin filaments is faint due to the low percentage of Cy5-labeled actin and low laser intensity used to prevent camera pixel saturation by the bright actin filament bundles. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.026
© Copyright Policy
Related In: Results  -  Collection

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

fig8s1: 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-Lpd850−1250aa or (B) 1 µM GFP-LZ-Lpd850−1250aa (dimer concentration equals 0.5 µM) in TIRF buffer containing 50 mM KCl. Note that the intensity of single actin filaments is faint due to the low percentage of Cy5-labeled actin and low laser intensity used to prevent camera pixel saturation by the bright actin filament bundles. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.026
Mentions: Because human VASP is a weakly processive actin polymerase and Lpd can interact simultaneously with both VASP tetramers and actin filaments, we hypothesized that Lpd might modulate the processivity of VASP by both clustering tetramers and tethering them to actin filaments. We tested this hypothesis by visualizing actin filament elongation in the presence of either monomeric (Lpd850−1250aa) or dimeric (LZ-Lpd850−1250aa) GFP-Lpd constructs. The weak interaction between monomeric GFP-Lpd850−1250aa and filamentous actin turns out to be further antagonized by the presence of monomeric actin or profilin-actin (Figure 8A). Also, we observed a reduction in the rate of barbed end actin filament elongation in the presence 2 µM profilin-actin and soluble GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa (Figure 8B), consistent with Lpd binding to (and partially sequestering) actin monomers. In contrast to monomeric Lpd, dimeric GFP-LZ-Lpd850−1250aa could transiently interact with both the sides and barbed ends of single growing actin filaments (Figure 8D, Video 8). Although GFP-Lpd and GFP-LZ-Lpd associate more transiently with single actin filaments in the presence of monomeric actin, Lpd binds well enough to promote filament bundling when present at a near stoichiometric concentration relative to monomeric actin (Figure 8—figure supplement 1).10.7554/eLife.06585.025Figure 8.Lpd enhances VASP barbed end processivity.

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