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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

Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).(A) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization. Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell body, following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B. Note that a population of GFP-Lpd850−1250aa remains associated with the peripheral membrane after addition of Jasp-LatB (yellow arrowhead). Horizontal scale bar, 5 µm. Vertical scale bar, 2 min. (B–E) Barbed ends are required for plasma membrane localization of (B) GFP-Lpd850−1250aa, (C) GFP-LZ-Lpd850−1250aa, (D) GFP-Lpd850−1250aa (AAPPP)x6, and (E) GFP-Lpd850−1250aa (AAPPPx6 + SH3*). Representative kymographs showing membrane dissociation of GFP-Lpd850−1250aa, wild-type and mutants, following the addition of 100 nM Cytochalasin D (blue arrowhead). Horizontal scale bar, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.019
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fig6: Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).(A) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization. Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell body, following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B. Note that a population of GFP-Lpd850−1250aa remains associated with the peripheral membrane after addition of Jasp-LatB (yellow arrowhead). Horizontal scale bar, 5 µm. Vertical scale bar, 2 min. (B–E) Barbed ends are required for plasma membrane localization of (B) GFP-Lpd850−1250aa, (C) GFP-LZ-Lpd850−1250aa, (D) GFP-Lpd850−1250aa (AAPPP)x6, and (E) GFP-Lpd850−1250aa (AAPPPx6 + SH3*). Representative kymographs showing membrane dissociation of GFP-Lpd850−1250aa, wild-type and mutants, following the addition of 100 nM Cytochalasin D (blue arrowhead). Horizontal scale bar, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.019

Mentions: To test whether leading edge membrane localization of GFP-Lpd850−1250aa requires interaction with free actin filament barbed ends or with the sides of actin filaments, we used pharmacological perturbations to examine how GFP-Lpd850−1250aa leading edge localization responds to changes in actin cytoskeletal dynamics. First, we simply froze actin assembly and disassembly by adding a mixture of Jasplakinolide and Latrunculin B to cells, without inhibiting myosin activity. Under these conditions we observed rapid retrograde movement of GFP-Lpd850−1250aa away from the leading edge, identical to the movement of mCherry-Actin (Figure 6A, Video 5). The dynamics of GFP-Lpd850−1250aa translocation suggests that, in the absence of net actin assembly, myosin can pull GFP-Lpd850−1250aa off the leading edge via a tight linkage to the actin cytoskeleton. In these experiments, a fraction of membrane-localized GFP-Lpd850−1250aa remained at the leading edge, presumably due to the maintenance of interaction with Ena/VASP and/or Abi1/endophilin.10.7554/eLife.06585.019Figure 6.Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).


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

Hansen SD, Mullins RD - Elife (2015)

Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).(A) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization. Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell body, following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B. Note that a population of GFP-Lpd850−1250aa remains associated with the peripheral membrane after addition of Jasp-LatB (yellow arrowhead). Horizontal scale bar, 5 µm. Vertical scale bar, 2 min. (B–E) Barbed ends are required for plasma membrane localization of (B) GFP-Lpd850−1250aa, (C) GFP-LZ-Lpd850−1250aa, (D) GFP-Lpd850−1250aa (AAPPP)x6, and (E) GFP-Lpd850−1250aa (AAPPPx6 + SH3*). Representative kymographs showing membrane dissociation of GFP-Lpd850−1250aa, wild-type and mutants, following the addition of 100 nM Cytochalasin D (blue arrowhead). Horizontal scale bar, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.019
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fig6: Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).(A) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization. Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell body, following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B. Note that a population of GFP-Lpd850−1250aa remains associated with the peripheral membrane after addition of Jasp-LatB (yellow arrowhead). Horizontal scale bar, 5 µm. Vertical scale bar, 2 min. (B–E) Barbed ends are required for plasma membrane localization of (B) GFP-Lpd850−1250aa, (C) GFP-LZ-Lpd850−1250aa, (D) GFP-Lpd850−1250aa (AAPPP)x6, and (E) GFP-Lpd850−1250aa (AAPPPx6 + SH3*). Representative kymographs showing membrane dissociation of GFP-Lpd850−1250aa, wild-type and mutants, following the addition of 100 nM Cytochalasin D (blue arrowhead). Horizontal scale bar, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.019
Mentions: To test whether leading edge membrane localization of GFP-Lpd850−1250aa requires interaction with free actin filament barbed ends or with the sides of actin filaments, we used pharmacological perturbations to examine how GFP-Lpd850−1250aa leading edge localization responds to changes in actin cytoskeletal dynamics. First, we simply froze actin assembly and disassembly by adding a mixture of Jasplakinolide and Latrunculin B to cells, without inhibiting myosin activity. Under these conditions we observed rapid retrograde movement of GFP-Lpd850−1250aa away from the leading edge, identical to the movement of mCherry-Actin (Figure 6A, Video 5). The dynamics of GFP-Lpd850−1250aa translocation suggests that, in the absence of net actin assembly, myosin can pull GFP-Lpd850−1250aa off the leading edge via a tight linkage to the actin cytoskeleton. In these experiments, a fraction of membrane-localized GFP-Lpd850−1250aa remained at the leading edge, presumably due to the maintenance of interaction with Ena/VASP and/or Abi1/endophilin.10.7554/eLife.06585.019Figure 6.Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd (850–1250aa).

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