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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.

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Schematic model of endoplasmic spreading. Initially the cell begins to spread via actin polymerization against the cell membrane. Cortical actin is cross-linked by Fln along the membrane. As the cell continues to spread, actin polymerization forces at the edge are transmitted back through the Fln-cross-linked cortex to the cell center, causing flattening of the endoplasm. At the same time, early adhesions begin to form. At the onset of contraction, cells exert contractile forces through mature focal adhesions, Fln-bundled actin stress fibers, and Fln-cross-linked actin meshworks (not shown for simplicity), allowing the endoplasm to spread in the direction of the edge despite being already flattened. Fln-depleted MEFs do not exhibit endoplasmic compression because they lack cortical cross-linking while they also do not experience contractile endoplasmic spreading due to unstable focal adhesions and a lack of strong actin stress fibers and cross-linked actin capable of stably supporting large forces exerted on the substrate.
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Figure 8: Schematic model of endoplasmic spreading. Initially the cell begins to spread via actin polymerization against the cell membrane. Cortical actin is cross-linked by Fln along the membrane. As the cell continues to spread, actin polymerization forces at the edge are transmitted back through the Fln-cross-linked cortex to the cell center, causing flattening of the endoplasm. At the same time, early adhesions begin to form. At the onset of contraction, cells exert contractile forces through mature focal adhesions, Fln-bundled actin stress fibers, and Fln-cross-linked actin meshworks (not shown for simplicity), allowing the endoplasm to spread in the direction of the edge despite being already flattened. Fln-depleted MEFs do not exhibit endoplasmic compression because they lack cortical cross-linking while they also do not experience contractile endoplasmic spreading due to unstable focal adhesions and a lack of strong actin stress fibers and cross-linked actin capable of stably supporting large forces exerted on the substrate.

Mentions: There appear to be two different roles for Fln in endoplasmic spreading (Figure 8). During the initial phases of spreading, the cortical cytoskeleton is flattened, which will cause the endoplasm to flatten as well. On the activation of contraction, the endoplasm will spread because of forces developed on the peripheral adhesions. In the absence of Fln, both mechanisms of spreading are weakened because of the loss of cross-links in the actin network. Connections between the endoplasm and surrounding cortical actin are particularly sensitive, explaining why gaps in the membrane form in this region in the FlnA–/– cells and why there are abrupt retractions of the endoplasmic boundary (Figure 6D). High forces generated within the contractile actin network surrounding the endoplasm that are normally transmitted through these connections would resist extension of MTs and the endoplasm boundary with the cell edge. This is still the case when calpain-uncleavable FlnA and FlnA Δ19–21 are added to the Fln-depleted system, underlining the importance of the Fln-adhesion linkage as well as the dynamic nature of actin cross-links (Figure 7).


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)

Schematic model of endoplasmic spreading. Initially the cell begins to spread via actin polymerization against the cell membrane. Cortical actin is cross-linked by Fln along the membrane. As the cell continues to spread, actin polymerization forces at the edge are transmitted back through the Fln-cross-linked cortex to the cell center, causing flattening of the endoplasm. At the same time, early adhesions begin to form. At the onset of contraction, cells exert contractile forces through mature focal adhesions, Fln-bundled actin stress fibers, and Fln-cross-linked actin meshworks (not shown for simplicity), allowing the endoplasm to spread in the direction of the edge despite being already flattened. Fln-depleted MEFs do not exhibit endoplasmic compression because they lack cortical cross-linking while they also do not experience contractile endoplasmic spreading due to unstable focal adhesions and a lack of strong actin stress fibers and cross-linked actin capable of stably supporting large forces exerted on the substrate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Schematic model of endoplasmic spreading. Initially the cell begins to spread via actin polymerization against the cell membrane. Cortical actin is cross-linked by Fln along the membrane. As the cell continues to spread, actin polymerization forces at the edge are transmitted back through the Fln-cross-linked cortex to the cell center, causing flattening of the endoplasm. At the same time, early adhesions begin to form. At the onset of contraction, cells exert contractile forces through mature focal adhesions, Fln-bundled actin stress fibers, and Fln-cross-linked actin meshworks (not shown for simplicity), allowing the endoplasm to spread in the direction of the edge despite being already flattened. Fln-depleted MEFs do not exhibit endoplasmic compression because they lack cortical cross-linking while they also do not experience contractile endoplasmic spreading due to unstable focal adhesions and a lack of strong actin stress fibers and cross-linked actin capable of stably supporting large forces exerted on the substrate.
Mentions: There appear to be two different roles for Fln in endoplasmic spreading (Figure 8). During the initial phases of spreading, the cortical cytoskeleton is flattened, which will cause the endoplasm to flatten as well. On the activation of contraction, the endoplasm will spread because of forces developed on the peripheral adhesions. In the absence of Fln, both mechanisms of spreading are weakened because of the loss of cross-links in the actin network. Connections between the endoplasm and surrounding cortical actin are particularly sensitive, explaining why gaps in the membrane form in this region in the FlnA–/– cells and why there are abrupt retractions of the endoplasmic boundary (Figure 6D). High forces generated within the contractile actin network surrounding the endoplasm that are normally transmitted through these connections would resist extension of MTs and the endoplasm boundary with the cell edge. This is still the case when calpain-uncleavable FlnA and FlnA Δ19–21 are added to the Fln-depleted system, underlining the importance of the Fln-adhesion linkage as well as the dynamic nature of actin cross-links (Figure 7).

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