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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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Calpain cleavability and integrin binding are FlnA functions critical in the ER spreading phenotype. Fln-depleted MEFs were transfected with RFP-ER and FL FlnA, calpain-uncleavable FlnA, or FlnA lacking domains 19–21. FL FlnA recovers the ER spreading phenotype to above control levels, whereas calpain-uncleavable FlnA and FlnA lacking domains 19–21 exhibit only partial recovery (n = 36) (***p < 0.001).
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Figure 7: Calpain cleavability and integrin binding are FlnA functions critical in the ER spreading phenotype. Fln-depleted MEFs were transfected with RFP-ER and FL FlnA, calpain-uncleavable FlnA, or FlnA lacking domains 19–21. FL FlnA recovers the ER spreading phenotype to above control levels, whereas calpain-uncleavable FlnA and FlnA lacking domains 19–21 exhibit only partial recovery (n = 36) (***p < 0.001).

Mentions: To further understand Fln’s role in the Fln-depleted endoplasmic spreading phenotype, Fln-depleted MEFs were transfected with RFP-ER and human full-length (FL) FlnA, calpain-uncleavable FlnA, or FlnA Δ19–21, the latter missing the primary integrin-binding site of FlnA. Normalized ER areas showed complete rescue of endoplasmic spreading upon FL FlnA addition, whereas calpain-uncleavable FlnA and FlnA Δ19–21 showed only partial rescue (Figure 7), indicating that both integrin binding and calpain cleavage play significant roles in Fln organization and function.


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)

Calpain cleavability and integrin binding are FlnA functions critical in the ER spreading phenotype. Fln-depleted MEFs were transfected with RFP-ER and FL FlnA, calpain-uncleavable FlnA, or FlnA lacking domains 19–21. FL FlnA recovers the ER spreading phenotype to above control levels, whereas calpain-uncleavable FlnA and FlnA lacking domains 19–21 exhibit only partial recovery (n = 36) (***p < 0.001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
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Figure 7: Calpain cleavability and integrin binding are FlnA functions critical in the ER spreading phenotype. Fln-depleted MEFs were transfected with RFP-ER and FL FlnA, calpain-uncleavable FlnA, or FlnA lacking domains 19–21. FL FlnA recovers the ER spreading phenotype to above control levels, whereas calpain-uncleavable FlnA and FlnA lacking domains 19–21 exhibit only partial recovery (n = 36) (***p < 0.001).
Mentions: To further understand Fln’s role in the Fln-depleted endoplasmic spreading phenotype, Fln-depleted MEFs were transfected with RFP-ER and human full-length (FL) FlnA, calpain-uncleavable FlnA, or FlnA Δ19–21, the latter missing the primary integrin-binding site of FlnA. Normalized ER areas showed complete rescue of endoplasmic spreading upon FL FlnA addition, whereas calpain-uncleavable FlnA and FlnA Δ19–21 showed only partial rescue (Figure 7), indicating that both integrin binding and calpain cleavage play significant roles in Fln organization and function.

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