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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

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) Representative kymographs showing retrograde flow of (C) monomeric GFP-Lpd850−1250aa and (D) dimeric GFP-LZ-Lpd850−1250aa. Compared to (A, B), images were acquired every 2 s. Scale bars, 5 µm and 1 min.DOI:http://dx.doi.org/10.7554/eLife.06585.010
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fig4s2: 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) Representative kymographs showing retrograde flow of (C) monomeric GFP-Lpd850−1250aa and (D) dimeric GFP-LZ-Lpd850−1250aa. Compared to (A, B), images were acquired every 2 s. Scale bars, 5 µm and 1 min.DOI:http://dx.doi.org/10.7554/eLife.06585.010

Mentions: To determine which Lpd binding partners—actin, Ena/VASP, Abi1/endophilin, inositol phospholipids, or Ras/Rho-family G-proteins—are required for leading edge localization, we compared the localization of various Lpd truncation mutants in XTC cells. We discovered that not only does GFP-Lpd850−1250aa, which lacks the tandem RA-PH domains, localize to leading edge membranes in XTC cells (Figure 4C), this construct also undergoes retrograde flow with the lamellipodial actin network. Analysis of speckle velocities in XTC cells co-expressing GFP-Lpd850−1250aa and mCherry-Actin revealed that the two proteins move with the same mean velocity: 71.9 ± 17.5 nm/s for Lpd850−1250aa vs 73.5 ± 14 nm/s for actin (Figure 4F, Video 2). In contrast to mCherry-Actin, GFP-Lpd850−1250aa speckles were observed less frequently, appeared more diffuse, and had shorter lifetimes (Figure 4—figure supplement 2). We suspect that the larger GFP-Lpd850−1250aa fluorescent particles are associated with fast endophilin-mediated endocytosis (FEME, Vehlow et al., 2013; Boucrot et al., 2015). Consistent with constitutively dimeric Lpd having a higher affinity for actin, GFP-LZ-Lpd850−1250aa displayed slightly more robust leading edge membrane localization and the intensity of presumptive endocytic particles were brighter, as compared to those observed in cells expressing GFP-Lpd850−1250aa (Figure 4—figure supplements 1, 2).Video 2.Retrograde flow of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells spread on poly-L-lysine (PLL) coated coverslips.


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

Hansen SD, Mullins RD - Elife (2015)

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) Representative kymographs showing retrograde flow of (C) monomeric GFP-Lpd850−1250aa and (D) dimeric GFP-LZ-Lpd850−1250aa. Compared to (A, B), images were acquired every 2 s. Scale bars, 5 µm and 1 min.DOI:http://dx.doi.org/10.7554/eLife.06585.010
© Copyright Policy
Related In: Results  -  Collection

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

fig4s2: 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) Representative kymographs showing retrograde flow of (C) monomeric GFP-Lpd850−1250aa and (D) dimeric GFP-LZ-Lpd850−1250aa. Compared to (A, B), images were acquired every 2 s. Scale bars, 5 µm and 1 min.DOI:http://dx.doi.org/10.7554/eLife.06585.010
Mentions: To determine which Lpd binding partners—actin, Ena/VASP, Abi1/endophilin, inositol phospholipids, or Ras/Rho-family G-proteins—are required for leading edge localization, we compared the localization of various Lpd truncation mutants in XTC cells. We discovered that not only does GFP-Lpd850−1250aa, which lacks the tandem RA-PH domains, localize to leading edge membranes in XTC cells (Figure 4C), this construct also undergoes retrograde flow with the lamellipodial actin network. Analysis of speckle velocities in XTC cells co-expressing GFP-Lpd850−1250aa and mCherry-Actin revealed that the two proteins move with the same mean velocity: 71.9 ± 17.5 nm/s for Lpd850−1250aa vs 73.5 ± 14 nm/s for actin (Figure 4F, Video 2). In contrast to mCherry-Actin, GFP-Lpd850−1250aa speckles were observed less frequently, appeared more diffuse, and had shorter lifetimes (Figure 4—figure supplement 2). We suspect that the larger GFP-Lpd850−1250aa fluorescent particles are associated with fast endophilin-mediated endocytosis (FEME, Vehlow et al., 2013; Boucrot et al., 2015). Consistent with constitutively dimeric Lpd having a higher affinity for actin, GFP-LZ-Lpd850−1250aa displayed slightly more robust leading edge membrane localization and the intensity of presumptive endocytic particles were brighter, as compared to those observed in cells expressing GFP-Lpd850−1250aa (Figure 4—figure supplements 1, 2).Video 2.Retrograde flow of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells spread on poly-L-lysine (PLL) coated coverslips.

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