Limits...
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

Localization of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa).(A, C) Representative images showing the plasma membrane localization of (A) GFP-Lpd1−1250aa, (B) GFP-Lpd850−1250aa, and (C) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells spread on PLL. Image in (A), marked with asterisk (*), is a representative cell imaged using with wide-field epifluorescence. Note the cytoplasmic localization of GFP-Lpd1−1250aa is not visible by TIRF microscopy. Scale bar, 20 µm. (D) Representative images of GFP-Lpd850−1250aa localization in polarized B16F1 cell migrating on laminin coated glass and imaged with wide-field epifluorescence. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.009
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543927&req=5

fig4s1: Localization of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa).(A, C) Representative images showing the plasma membrane localization of (A) GFP-Lpd1−1250aa, (B) GFP-Lpd850−1250aa, and (C) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells spread on PLL. Image in (A), marked with asterisk (*), is a representative cell imaged using with wide-field epifluorescence. Note the cytoplasmic localization of GFP-Lpd1−1250aa is not visible by TIRF microscopy. Scale bar, 20 µm. (D) Representative images of GFP-Lpd850−1250aa localization in polarized B16F1 cell migrating on laminin coated glass and imaged with wide-field epifluorescence. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.009

Mentions: Having found that Lpd binds actin filaments in vitro, we next worked to determine whether actin binding by Lpd plays a significant role in its cellular localization and function. To address this question we visualized the localization of fluorescently tagged, FL Lpd (GFP-Lpd1−1250aa) in Xenopus Tissue Culture (XTC) cells, spread on poly-L-lysine (PLL)-coated coverslips (Figure 4A). We used XTC cells because they spread well and produce very thin peripheral actin networks, well suited for fluorescence microscopy. Ectopic gene expression from a truncated cytomegalovirus (CMV) promoter produces very low levels of protein expression in XTC cells, ideal for single-molecule fluorescence microscopy (Watanabe and Mitchison, 2002). TIRF microscopy imaging of single GFP-Lpd1−1250aa molecules revealed that FL Lpd localizes predominantly to the leading edge of ruffling membranes and cycles on and off the plasma membrane on a subsecond time scale (Figure 4B, Figure 4—figure supplement 1, Video 1). Leading edge membrane localization of GFP-Lpd1−1250aa required dynamic actin assembly and disassembly, because acute treatment of cells with a chemical inhibitor cocktail that freezes actin dynamics (JLY drug cocktail: Jasplakinolide, Latrunculin B, and Y27632 Rock kinase inhibitor [Peng et al., 2011]) caused rapid loss of membrane-localized Lpd (Figure 4C).10.7554/eLife.06585.008Figure 4.Lpd (850–1250aa) localizes to the leading edge membranes and undergoes retrograde flow with the actin cytoskeleton.


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

Hansen SD, Mullins RD - Elife (2015)

Localization of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa).(A, C) Representative images showing the plasma membrane localization of (A) GFP-Lpd1−1250aa, (B) GFP-Lpd850−1250aa, and (C) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells spread on PLL. Image in (A), marked with asterisk (*), is a representative cell imaged using with wide-field epifluorescence. Note the cytoplasmic localization of GFP-Lpd1−1250aa is not visible by TIRF microscopy. Scale bar, 20 µm. (D) Representative images of GFP-Lpd850−1250aa localization in polarized B16F1 cell migrating on laminin coated glass and imaged with wide-field epifluorescence. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.009
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: Localization of GFP-Lpd (850–1250aa) and GFP-LZ-Lpd (850–1250aa).(A, C) Representative images showing the plasma membrane localization of (A) GFP-Lpd1−1250aa, (B) GFP-Lpd850−1250aa, and (C) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells spread on PLL. Image in (A), marked with asterisk (*), is a representative cell imaged using with wide-field epifluorescence. Note the cytoplasmic localization of GFP-Lpd1−1250aa is not visible by TIRF microscopy. Scale bar, 20 µm. (D) Representative images of GFP-Lpd850−1250aa localization in polarized B16F1 cell migrating on laminin coated glass and imaged with wide-field epifluorescence. Scale bar, 20 µm.DOI:http://dx.doi.org/10.7554/eLife.06585.009
Mentions: Having found that Lpd binds actin filaments in vitro, we next worked to determine whether actin binding by Lpd plays a significant role in its cellular localization and function. To address this question we visualized the localization of fluorescently tagged, FL Lpd (GFP-Lpd1−1250aa) in Xenopus Tissue Culture (XTC) cells, spread on poly-L-lysine (PLL)-coated coverslips (Figure 4A). We used XTC cells because they spread well and produce very thin peripheral actin networks, well suited for fluorescence microscopy. Ectopic gene expression from a truncated cytomegalovirus (CMV) promoter produces very low levels of protein expression in XTC cells, ideal for single-molecule fluorescence microscopy (Watanabe and Mitchison, 2002). TIRF microscopy imaging of single GFP-Lpd1−1250aa molecules revealed that FL Lpd localizes predominantly to the leading edge of ruffling membranes and cycles on and off the plasma membrane on a subsecond time scale (Figure 4B, Figure 4—figure supplement 1, Video 1). Leading edge membrane localization of GFP-Lpd1−1250aa required dynamic actin assembly and disassembly, because acute treatment of cells with a chemical inhibitor cocktail that freezes actin dynamics (JLY drug cocktail: Jasplakinolide, Latrunculin B, and Y27632 Rock kinase inhibitor [Peng et al., 2011]) caused rapid loss of membrane-localized Lpd (Figure 4C).10.7554/eLife.06585.008Figure 4.Lpd (850–1250aa) localizes to the leading edge membranes and undergoes retrograde flow with the actin cytoskeleton.

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