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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

Actin based motility on lipid coated glass beads.(A) Montage of actin comet tails frozen at different times point with 37.5 µM Latrunculin B-phalloidin containing buffer. Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), 50 nM Arp2/3, 100 nM capping protein, 6 µM hPro1, 3 µM cofilin. Scale bar, 10 µm. (B) Image of actin comet tails at steady-state in the presence of cofilin dependent network recycling described in (A). Scale bar, 10 µm. (C) Images of asymmetric his10-Cherry-SCARAPWCA localization in the presence of actin comet tail. Scale bar, 5 µm. (D) Line scan across lipid coated bead surface in (C) showing fluorescent intensity of asymmetric his10-Cherry-SCARAPWCA.DOI:http://dx.doi.org/10.7554/eLife.06585.007
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fig3s1: Actin based motility on lipid coated glass beads.(A) Montage of actin comet tails frozen at different times point with 37.5 µM Latrunculin B-phalloidin containing buffer. Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), 50 nM Arp2/3, 100 nM capping protein, 6 µM hPro1, 3 µM cofilin. Scale bar, 10 µm. (B) Image of actin comet tails at steady-state in the presence of cofilin dependent network recycling described in (A). Scale bar, 10 µm. (C) Images of asymmetric his10-Cherry-SCARAPWCA localization in the presence of actin comet tail. Scale bar, 5 µm. (D) Line scan across lipid coated bead surface in (C) showing fluorescent intensity of asymmetric his10-Cherry-SCARAPWCA.DOI:http://dx.doi.org/10.7554/eLife.06585.007

Mentions: We next tested whether Lpd can interact directly with artificial lamellipodial actin networks reconstituted in vitro from purified components (Loisel et al., 1999; Akin and Mullins, 2008). We used the Arp2/3 complex, together with capping protein, to assemble dendritic actin networks on lipid-coated bead (LCBs) containing Ni-conjugated lipids bound to his10Cherry-SCAR. In these assays we also included either membrane tethered his10GFP or his10GFP-Lpd850−1250aa (Figure 3A, Figure 3—figure supplement 1). Since his10GFP-Lpd850−1250aa is tethered to the membrane via Ni-conjugated lipids, our reconstitution lacks the recruitment and dissociation dynamics that may be mediated by interactions between the Lpd PH domain and phosphatidylinositol lipids (i.e., PI(3,4)P2). Nonetheless, we used this assay to test whether membrane tethered Lpd modulates actin network assembly in an autonomous manner. By measuring the length and density of actin comet tails at various times (Figure 3B), we found that membrane-tethered Lpd significantly slowed the rate of actin network assembly (1.5 ± 0.25 µm/min) compared to membrane-tethered GFP alone (7.0 ± 0.48 µm/min; Figure 3B,C).10.7554/eLife.06585.006Figure 3.Membrane-tethered Lpd slows dendritic actin network assembly in vitro.


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

Hansen SD, Mullins RD - Elife (2015)

Actin based motility on lipid coated glass beads.(A) Montage of actin comet tails frozen at different times point with 37.5 µM Latrunculin B-phalloidin containing buffer. Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), 50 nM Arp2/3, 100 nM capping protein, 6 µM hPro1, 3 µM cofilin. Scale bar, 10 µm. (B) Image of actin comet tails at steady-state in the presence of cofilin dependent network recycling described in (A). Scale bar, 10 µm. (C) Images of asymmetric his10-Cherry-SCARAPWCA localization in the presence of actin comet tail. Scale bar, 5 µm. (D) Line scan across lipid coated bead surface in (C) showing fluorescent intensity of asymmetric his10-Cherry-SCARAPWCA.DOI:http://dx.doi.org/10.7554/eLife.06585.007
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Actin based motility on lipid coated glass beads.(A) Montage of actin comet tails frozen at different times point with 37.5 µM Latrunculin B-phalloidin containing buffer. Actin networks were assembled in the presence of 7.5 µM actin (5% Alexa488), 50 nM Arp2/3, 100 nM capping protein, 6 µM hPro1, 3 µM cofilin. Scale bar, 10 µm. (B) Image of actin comet tails at steady-state in the presence of cofilin dependent network recycling described in (A). Scale bar, 10 µm. (C) Images of asymmetric his10-Cherry-SCARAPWCA localization in the presence of actin comet tail. Scale bar, 5 µm. (D) Line scan across lipid coated bead surface in (C) showing fluorescent intensity of asymmetric his10-Cherry-SCARAPWCA.DOI:http://dx.doi.org/10.7554/eLife.06585.007
Mentions: We next tested whether Lpd can interact directly with artificial lamellipodial actin networks reconstituted in vitro from purified components (Loisel et al., 1999; Akin and Mullins, 2008). We used the Arp2/3 complex, together with capping protein, to assemble dendritic actin networks on lipid-coated bead (LCBs) containing Ni-conjugated lipids bound to his10Cherry-SCAR. In these assays we also included either membrane tethered his10GFP or his10GFP-Lpd850−1250aa (Figure 3A, Figure 3—figure supplement 1). Since his10GFP-Lpd850−1250aa is tethered to the membrane via Ni-conjugated lipids, our reconstitution lacks the recruitment and dissociation dynamics that may be mediated by interactions between the Lpd PH domain and phosphatidylinositol lipids (i.e., PI(3,4)P2). Nonetheless, we used this assay to test whether membrane tethered Lpd modulates actin network assembly in an autonomous manner. By measuring the length and density of actin comet tails at various times (Figure 3B), we found that membrane-tethered Lpd significantly slowed the rate of actin network assembly (1.5 ± 0.25 µm/min) compared to membrane-tethered GFP alone (7.0 ± 0.48 µm/min; Figure 3B,C).10.7554/eLife.06585.006Figure 3.Membrane-tethered Lpd slows dendritic actin network assembly 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