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Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.

Oakes PW, Beckham Y, Stricker J, Gardel ML - J. Cell Biol. (2012)

Bottom Line: As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown.We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions.We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.

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Suppressing RSF assembly reduces the quantity of active myosin II but increases lamellar actin retrograde flow rate. (A) Immunofluorescence images of pMLC and fluorescent phalloidin staining of F-actin are shown for WT, Dia Inh, and Atn-1 KD U2OS cells. Bar, 20 µm. (B) Western blots showing MLC and pMLC for each condition in relation to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. (C) Densitometry analysis of Western blots showing the relative levels of pMLC, MLC, and the ratio of pMLC to MLC for each condition, normalized to WT cells. There is no significant difference in the ratio of MLC to pMLC for each condition. The bar plot shows the mean ratio relative to WT cells (error bars indicate SEM; n = 6 for each condition). (D) A series of images of GFP-actin expressed in a protruding Atn-1 KD cell over time. Time is indicated in minutes/seconds. Bar, 10 µm. See Video 1. (E) Magnified images of the boxed region indicated at time 0 in D, illustrating the reorientation of a transverse arc to align in the direction of retrograde flow (red arrowheads). Bar, 2 µm. (F) Actin flow vectors are overlaid on the GFP-actin image from E at 4 min and 30 s. The white arrow represents 15 nm/s. Bar, 10 µm. (G) A box plot of the lamellar retrograde flow speed for each condition described (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 150 flow vectors from multiple regions in three to five cells for each condition; ***, P < 0.001 with respect to WT).
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fig2: Suppressing RSF assembly reduces the quantity of active myosin II but increases lamellar actin retrograde flow rate. (A) Immunofluorescence images of pMLC and fluorescent phalloidin staining of F-actin are shown for WT, Dia Inh, and Atn-1 KD U2OS cells. Bar, 20 µm. (B) Western blots showing MLC and pMLC for each condition in relation to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. (C) Densitometry analysis of Western blots showing the relative levels of pMLC, MLC, and the ratio of pMLC to MLC for each condition, normalized to WT cells. There is no significant difference in the ratio of MLC to pMLC for each condition. The bar plot shows the mean ratio relative to WT cells (error bars indicate SEM; n = 6 for each condition). (D) A series of images of GFP-actin expressed in a protruding Atn-1 KD cell over time. Time is indicated in minutes/seconds. Bar, 10 µm. See Video 1. (E) Magnified images of the boxed region indicated at time 0 in D, illustrating the reorientation of a transverse arc to align in the direction of retrograde flow (red arrowheads). Bar, 2 µm. (F) Actin flow vectors are overlaid on the GFP-actin image from E at 4 min and 30 s. The white arrow represents 15 nm/s. Bar, 10 µm. (G) A box plot of the lamellar retrograde flow speed for each condition described (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 150 flow vectors from multiple regions in three to five cells for each condition; ***, P < 0.001 with respect to WT).

Mentions: To explore the extent to which the organization of active myosin II within the lamella was affected by the inhibition of RSF assembly, we visualized the enzymatically active fraction of myosin II by immunofluorescence of myosin light chain (MLC) phosphorylated at serine 19 (pMLC; Adelstein and Conti, 1975) while also visualizing the F-actin cytoskeleton with fluorescent phalloidin (Fig. 2 A). In all populations, active myosin localized throughout the lamella primarily along transverse arcs (Fig. 2 A). We tested cell lysates for relative amounts of pMLC and total MLC via Western blotting (Fig. 2 B) and used densitometry to quantify the ratio of pMLC to MLC (Fig. 2 C). We found small reductions in the amount of MLC and pMLC but found no significant difference in the ratio of pMLC to MLC in both Dia-inhibited and Atn-1 KD cells. We speculate that this small reduction in active myosin may be a result of changes in biochemical signaling to Rho pathways caused by changes in lamellar organization or focal adhesions (Schwartz, 2010), but a definitive understanding would require further study.


Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.

Oakes PW, Beckham Y, Stricker J, Gardel ML - J. Cell Biol. (2012)

Suppressing RSF assembly reduces the quantity of active myosin II but increases lamellar actin retrograde flow rate. (A) Immunofluorescence images of pMLC and fluorescent phalloidin staining of F-actin are shown for WT, Dia Inh, and Atn-1 KD U2OS cells. Bar, 20 µm. (B) Western blots showing MLC and pMLC for each condition in relation to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. (C) Densitometry analysis of Western blots showing the relative levels of pMLC, MLC, and the ratio of pMLC to MLC for each condition, normalized to WT cells. There is no significant difference in the ratio of MLC to pMLC for each condition. The bar plot shows the mean ratio relative to WT cells (error bars indicate SEM; n = 6 for each condition). (D) A series of images of GFP-actin expressed in a protruding Atn-1 KD cell over time. Time is indicated in minutes/seconds. Bar, 10 µm. See Video 1. (E) Magnified images of the boxed region indicated at time 0 in D, illustrating the reorientation of a transverse arc to align in the direction of retrograde flow (red arrowheads). Bar, 2 µm. (F) Actin flow vectors are overlaid on the GFP-actin image from E at 4 min and 30 s. The white arrow represents 15 nm/s. Bar, 10 µm. (G) A box plot of the lamellar retrograde flow speed for each condition described (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 150 flow vectors from multiple regions in three to five cells for each condition; ***, P < 0.001 with respect to WT).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275371&req=5

fig2: Suppressing RSF assembly reduces the quantity of active myosin II but increases lamellar actin retrograde flow rate. (A) Immunofluorescence images of pMLC and fluorescent phalloidin staining of F-actin are shown for WT, Dia Inh, and Atn-1 KD U2OS cells. Bar, 20 µm. (B) Western blots showing MLC and pMLC for each condition in relation to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. (C) Densitometry analysis of Western blots showing the relative levels of pMLC, MLC, and the ratio of pMLC to MLC for each condition, normalized to WT cells. There is no significant difference in the ratio of MLC to pMLC for each condition. The bar plot shows the mean ratio relative to WT cells (error bars indicate SEM; n = 6 for each condition). (D) A series of images of GFP-actin expressed in a protruding Atn-1 KD cell over time. Time is indicated in minutes/seconds. Bar, 10 µm. See Video 1. (E) Magnified images of the boxed region indicated at time 0 in D, illustrating the reorientation of a transverse arc to align in the direction of retrograde flow (red arrowheads). Bar, 2 µm. (F) Actin flow vectors are overlaid on the GFP-actin image from E at 4 min and 30 s. The white arrow represents 15 nm/s. Bar, 10 µm. (G) A box plot of the lamellar retrograde flow speed for each condition described (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 150 flow vectors from multiple regions in three to five cells for each condition; ***, P < 0.001 with respect to WT).
Mentions: To explore the extent to which the organization of active myosin II within the lamella was affected by the inhibition of RSF assembly, we visualized the enzymatically active fraction of myosin II by immunofluorescence of myosin light chain (MLC) phosphorylated at serine 19 (pMLC; Adelstein and Conti, 1975) while also visualizing the F-actin cytoskeleton with fluorescent phalloidin (Fig. 2 A). In all populations, active myosin localized throughout the lamella primarily along transverse arcs (Fig. 2 A). We tested cell lysates for relative amounts of pMLC and total MLC via Western blotting (Fig. 2 B) and used densitometry to quantify the ratio of pMLC to MLC (Fig. 2 C). We found small reductions in the amount of MLC and pMLC but found no significant difference in the ratio of pMLC to MLC in both Dia-inhibited and Atn-1 KD cells. We speculate that this small reduction in active myosin may be a result of changes in biochemical signaling to Rho pathways caused by changes in lamellar organization or focal adhesions (Schwartz, 2010), but a definitive understanding would require further study.

Bottom Line: As myosin II activity drives stress fiber assembly and enhanced tension at adhesions simultaneously, the extent to which adhesion maturation is driven by tension or altered actin architecture is unknown.We show that perturbations to formin and α-actinin 1 activity selectively inhibited stress fiber assembly at adhesions but retained a contractile lamella that generated large tension on adhesions.We propose that stress fiber assembly at the adhesion site serves as a structural template that facilitates adhesion maturation over a wide range of tensions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA.

Show MeSH
Related in: MedlinePlus