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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

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 homology 1 (EVH1) BDs (i.e., FPPPP or LPPPP), while red boxes highlight the predicted Abi1/endophilin SH3 domain binding sites (i.e., PxxPxR). Secondary structure prediction algorithms suggest that the Lpd (850–1250aa) lacks secondary structure (data not shown). (B) Comparison of Lpd, Pico, mig-10, RIAM, ActA, and Zyxin pIs across the canonical Ena/VASP BD containing one or more FPPPP motifs. Domain boundaries for this region are termed, Lpd C-terminus (Lpd CT). The number of arginine and lysine residues were calculated across the region specified ‘Lpd C-terminus (Lpd CT)’. The number of Ena/VASP and SH3 domain binding sites were counted across the domain boundaries defined, Lpd CT, and are shown in columns five and six, respectively. The pIs for Lpd CT were calculated using EXPASY (Wilkins et al., 1999). Abbreviations are as follows: full-length (FL); binding-domain (BD); Lamellipodin (Lpd).DOI:http://dx.doi.org/10.7554/eLife.06585.005
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fig2: 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 homology 1 (EVH1) BDs (i.e., FPPPP or LPPPP), while red boxes highlight the predicted Abi1/endophilin SH3 domain binding sites (i.e., PxxPxR). Secondary structure prediction algorithms suggest that the Lpd (850–1250aa) lacks secondary structure (data not shown). (B) Comparison of Lpd, Pico, mig-10, RIAM, ActA, and Zyxin pIs across the canonical Ena/VASP BD containing one or more FPPPP motifs. Domain boundaries for this region are termed, Lpd C-terminus (Lpd CT). The number of arginine and lysine residues were calculated across the region specified ‘Lpd C-terminus (Lpd CT)’. The number of Ena/VASP and SH3 domain binding sites were counted across the domain boundaries defined, Lpd CT, and are shown in columns five and six, respectively. The pIs for Lpd CT were calculated using EXPASY (Wilkins et al., 1999). Abbreviations are as follows: full-length (FL); binding-domain (BD); Lamellipodin (Lpd).DOI:http://dx.doi.org/10.7554/eLife.06585.005

Mentions: Conserved acidic residues near the amino terminus of actin create an electronegative patch on the surface of actin filaments that interacts with positively charged, basic residues in many actin binding proteins (Fujii et al., 2010). The carboxy-terminal region of human Lpd (residues 850–1250) is highly basic, with an isoelectric point (pI) of 9.97 (Figure 2A,B). The distribution of basic residues in this region is evolutionarily conserved among Lpd homologs, but is not conserved in other Ena/VASP-binding proteins, such as Rap1-GTP-interacting adaptor molecule (RIAM), zyxin, or ActA (Figure 2B). Mutating all 44 basic amino acid residues in the C-terminal ABR of Lpd to alanines (to create Lpd(44A)850−1250aa) abolished filament binding (Figure 1D), but had no effect on the ability of this construct to bind to Ena/VASP proteins (unpublished results). Because the C-terminal region of Lpd appears to be natively unfolded, the loss of actin binding likely reflects simply a loss of electrostatic interactions rather than a change in protein structure. Furthermore, bacterially expressed and purified GFP-Lpd(44A)850−1250aa showed no signs of being structurally or functionally compromised, as compared wild-type GFP-Lpd850−1250aa.10.7554/eLife.06585.005Figure 2.Conservation of Lpd (850–1250aa) amino acid sequence and isoelectric point (pI).


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

Hansen SD, Mullins RD - Elife (2015)

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 homology 1 (EVH1) BDs (i.e., FPPPP or LPPPP), while red boxes highlight the predicted Abi1/endophilin SH3 domain binding sites (i.e., PxxPxR). Secondary structure prediction algorithms suggest that the Lpd (850–1250aa) lacks secondary structure (data not shown). (B) Comparison of Lpd, Pico, mig-10, RIAM, ActA, and Zyxin pIs across the canonical Ena/VASP BD containing one or more FPPPP motifs. Domain boundaries for this region are termed, Lpd C-terminus (Lpd CT). The number of arginine and lysine residues were calculated across the region specified ‘Lpd C-terminus (Lpd CT)’. The number of Ena/VASP and SH3 domain binding sites were counted across the domain boundaries defined, Lpd CT, and are shown in columns five and six, respectively. The pIs for Lpd CT were calculated using EXPASY (Wilkins et al., 1999). Abbreviations are as follows: full-length (FL); binding-domain (BD); Lamellipodin (Lpd).DOI:http://dx.doi.org/10.7554/eLife.06585.005
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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 homology 1 (EVH1) BDs (i.e., FPPPP or LPPPP), while red boxes highlight the predicted Abi1/endophilin SH3 domain binding sites (i.e., PxxPxR). Secondary structure prediction algorithms suggest that the Lpd (850–1250aa) lacks secondary structure (data not shown). (B) Comparison of Lpd, Pico, mig-10, RIAM, ActA, and Zyxin pIs across the canonical Ena/VASP BD containing one or more FPPPP motifs. Domain boundaries for this region are termed, Lpd C-terminus (Lpd CT). The number of arginine and lysine residues were calculated across the region specified ‘Lpd C-terminus (Lpd CT)’. The number of Ena/VASP and SH3 domain binding sites were counted across the domain boundaries defined, Lpd CT, and are shown in columns five and six, respectively. The pIs for Lpd CT were calculated using EXPASY (Wilkins et al., 1999). Abbreviations are as follows: full-length (FL); binding-domain (BD); Lamellipodin (Lpd).DOI:http://dx.doi.org/10.7554/eLife.06585.005
Mentions: Conserved acidic residues near the amino terminus of actin create an electronegative patch on the surface of actin filaments that interacts with positively charged, basic residues in many actin binding proteins (Fujii et al., 2010). The carboxy-terminal region of human Lpd (residues 850–1250) is highly basic, with an isoelectric point (pI) of 9.97 (Figure 2A,B). The distribution of basic residues in this region is evolutionarily conserved among Lpd homologs, but is not conserved in other Ena/VASP-binding proteins, such as Rap1-GTP-interacting adaptor molecule (RIAM), zyxin, or ActA (Figure 2B). Mutating all 44 basic amino acid residues in the C-terminal ABR of Lpd to alanines (to create Lpd(44A)850−1250aa) abolished filament binding (Figure 1D), but had no effect on the ability of this construct to bind to Ena/VASP proteins (unpublished results). Because the C-terminal region of Lpd appears to be natively unfolded, the loss of actin binding likely reflects simply a loss of electrostatic interactions rather than a change in protein structure. Furthermore, bacterially expressed and purified GFP-Lpd(44A)850−1250aa showed no signs of being structurally or functionally compromised, as compared wild-type GFP-Lpd850−1250aa.10.7554/eLife.06585.005Figure 2.Conservation of Lpd (850–1250aa) amino acid sequence and isoelectric point (pI).

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