Limits...
Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions.

Lynch CD, Gauthier NC, Biais N, Lazar AM, Roca-Cusachs P, Yu CH, Sheetz MP - Mol. Biol. Cell (2011)

Bottom Line: Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules.Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion.Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Show MeSH

Related in: MedlinePlus

Fln-depleted MEFs exhibit an inability to sustain large, controlled forces on FN-coated pillars. (A and B) Fln-depleted MEFs and controls were spread on FN-coated PDMS pillars for ∼30 min. Scale = 20 μm. (C and D) Representative traces of single pillar movements tracked over ∼30 min of spreading in control (C) and Fln-depleted (D) MEFs (traces taken from pillars marked by a red dot in A and B). (E) Colored regions from C and D reveal rapid force release in Fln-depleted MEFs compared with controls. (F) Average rate of force-release events on pillars. Fln-depleted MEFs exhibited a significantly higher rate of force release compared with controls (5.42 pN/s ± 1.50 pN/s compared with 1.36 pN/s ± 0.21 pN/s, n = 29, ***p < 0.001). (G) Mean force/pillar of Fln-depleted MEFs and controls shows no significant difference in force generation capability (n = 12; >3 experiments).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198308&req=5

Figure 5: Fln-depleted MEFs exhibit an inability to sustain large, controlled forces on FN-coated pillars. (A and B) Fln-depleted MEFs and controls were spread on FN-coated PDMS pillars for ∼30 min. Scale = 20 μm. (C and D) Representative traces of single pillar movements tracked over ∼30 min of spreading in control (C) and Fln-depleted (D) MEFs (traces taken from pillars marked by a red dot in A and B). (E) Colored regions from C and D reveal rapid force release in Fln-depleted MEFs compared with controls. (F) Average rate of force-release events on pillars. Fln-depleted MEFs exhibited a significantly higher rate of force release compared with controls (5.42 pN/s ± 1.50 pN/s compared with 1.36 pN/s ± 0.21 pN/s, n = 29, ***p < 0.001). (G) Mean force/pillar of Fln-depleted MEFs and controls shows no significant difference in force generation capability (n = 12; >3 experiments).

Mentions: Because unstable focal adhesions could result in defects of force generation, we measured the traction forces of Fln-depleted MEFs using an array of polydimethylsiloxane (PDMS) pillars (Tan et al., 2003; du Roure et al., 2005). When Fln-depleted MEFs were spread and imaged on PDMS pillars for ∼30 min, they exhibited more narrow extensions than did controls (Figure 5, A and B). Representative force-versus-time traces from single-pillar measurements exhibited similar or higher levels of peak force generation (Figure 5, C and D); however, the release of force occurred at a higher rate in Fln-depleted cells when compared with controls (Figure 5E). In Fln-depleted cells, the average rate of force release was nearly four times that of controls (Figure 5F). Because this behavior could result from impaired traction force generation, we measured whole-cell force generation and found a slightly lower traction force per pillar in Fln-depleted MEFs, but the average value was not significantly different from controls (Figure 5G).


Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions.

Lynch CD, Gauthier NC, Biais N, Lazar AM, Roca-Cusachs P, Yu CH, Sheetz MP - Mol. Biol. Cell (2011)

Fln-depleted MEFs exhibit an inability to sustain large, controlled forces on FN-coated pillars. (A and B) Fln-depleted MEFs and controls were spread on FN-coated PDMS pillars for ∼30 min. Scale = 20 μm. (C and D) Representative traces of single pillar movements tracked over ∼30 min of spreading in control (C) and Fln-depleted (D) MEFs (traces taken from pillars marked by a red dot in A and B). (E) Colored regions from C and D reveal rapid force release in Fln-depleted MEFs compared with controls. (F) Average rate of force-release events on pillars. Fln-depleted MEFs exhibited a significantly higher rate of force release compared with controls (5.42 pN/s ± 1.50 pN/s compared with 1.36 pN/s ± 0.21 pN/s, n = 29, ***p < 0.001). (G) Mean force/pillar of Fln-depleted MEFs and controls shows no significant difference in force generation capability (n = 12; >3 experiments).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198308&req=5

Figure 5: Fln-depleted MEFs exhibit an inability to sustain large, controlled forces on FN-coated pillars. (A and B) Fln-depleted MEFs and controls were spread on FN-coated PDMS pillars for ∼30 min. Scale = 20 μm. (C and D) Representative traces of single pillar movements tracked over ∼30 min of spreading in control (C) and Fln-depleted (D) MEFs (traces taken from pillars marked by a red dot in A and B). (E) Colored regions from C and D reveal rapid force release in Fln-depleted MEFs compared with controls. (F) Average rate of force-release events on pillars. Fln-depleted MEFs exhibited a significantly higher rate of force release compared with controls (5.42 pN/s ± 1.50 pN/s compared with 1.36 pN/s ± 0.21 pN/s, n = 29, ***p < 0.001). (G) Mean force/pillar of Fln-depleted MEFs and controls shows no significant difference in force generation capability (n = 12; >3 experiments).
Mentions: Because unstable focal adhesions could result in defects of force generation, we measured the traction forces of Fln-depleted MEFs using an array of polydimethylsiloxane (PDMS) pillars (Tan et al., 2003; du Roure et al., 2005). When Fln-depleted MEFs were spread and imaged on PDMS pillars for ∼30 min, they exhibited more narrow extensions than did controls (Figure 5, A and B). Representative force-versus-time traces from single-pillar measurements exhibited similar or higher levels of peak force generation (Figure 5, C and D); however, the release of force occurred at a higher rate in Fln-depleted cells when compared with controls (Figure 5E). In Fln-depleted cells, the average rate of force release was nearly four times that of controls (Figure 5F). Because this behavior could result from impaired traction force generation, we measured whole-cell force generation and found a slightly lower traction force per pillar in Fln-depleted MEFs, but the average value was not significantly different from controls (Figure 5G).

Bottom Line: Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules.Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion.Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Show MeSH
Related in: MedlinePlus