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 depletion causes adhesion defects. (A) Merged confocal sections of FlnB+/+ MEF 30 min after plating on FN-coated glass. Scale = 20 μm; green = paxillin, red = phalloidin, blue = FlnA). Enlarged regions highlight areas of FlnA enrichment, including large nodes at stress fiber junctions, the cell edge, adhesions, and transverse arcs surrounding the endoplasm. (B) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for paxillin and phalloidin. GFP-expressing cells were considered transfected and therefore Fln-depleted. Scale = 50 μm. (C) Merged images of paxillin and F-actin panels show that Fln-depleted MEFs exhibited a highly disorganized, stress fiber-free, actin meshwork surrounding the endoplasm as well as a lack of focal adhesions compared with nontransfected cells. Scale = 50 μm; green pseudocoloring = paxillin, magenta = phalloidin). (D) FlnB–/– MEFs were transfected with nontargeting or FlnA-targeting shRNA expression vectors, sorted by FACS, and lysed. GFP-expressing cells from these populations exhibit no difference in beta 1 integrin expression. (E) Fln-depleted MEFs and controls expressing paxillin-RFP were spread on FN-coated glass and imaged with TIRF microscopy. Scale = 5 μm. (F) Kymographs taken along white lines in E show that focal adhesions do not retain their stability over the course of spreading in Fln-depleted MEFs. Scale = 5 μm; 3 min. (G) Fln-depleted MEFs and controls were trypsinized, resuspended in 10 μM nocodazole in serum-free DMEM for 30 min, and allowed to spread. Red arrows indicate protrusive regions that collapse in subsequent frames. Scale = 20 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198308&req=5

Figure 4: Fln depletion causes adhesion defects. (A) Merged confocal sections of FlnB+/+ MEF 30 min after plating on FN-coated glass. Scale = 20 μm; green = paxillin, red = phalloidin, blue = FlnA). Enlarged regions highlight areas of FlnA enrichment, including large nodes at stress fiber junctions, the cell edge, adhesions, and transverse arcs surrounding the endoplasm. (B) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for paxillin and phalloidin. GFP-expressing cells were considered transfected and therefore Fln-depleted. Scale = 50 μm. (C) Merged images of paxillin and F-actin panels show that Fln-depleted MEFs exhibited a highly disorganized, stress fiber-free, actin meshwork surrounding the endoplasm as well as a lack of focal adhesions compared with nontransfected cells. Scale = 50 μm; green pseudocoloring = paxillin, magenta = phalloidin). (D) FlnB–/– MEFs were transfected with nontargeting or FlnA-targeting shRNA expression vectors, sorted by FACS, and lysed. GFP-expressing cells from these populations exhibit no difference in beta 1 integrin expression. (E) Fln-depleted MEFs and controls expressing paxillin-RFP were spread on FN-coated glass and imaged with TIRF microscopy. Scale = 5 μm. (F) Kymographs taken along white lines in E show that focal adhesions do not retain their stability over the course of spreading in Fln-depleted MEFs. Scale = 5 μm; 3 min. (G) Fln-depleted MEFs and controls were trypsinized, resuspended in 10 μM nocodazole in serum-free DMEM for 30 min, and allowed to spread. Red arrows indicate protrusive regions that collapse in subsequent frames. Scale = 20 μm.

Mentions: MT targeting is believed to be involved in focal adhesion dynamics (Kaverina et al., 1999; Krylyshkina et al., 2002, 2003). Because MTs do not extend to the periphery in Fln-depleted cells, peripheral focal adhesions may also be altered. To test this possibility, Fln-depleted MEFs and untransfected controls were spread for 1 h and stained with anti-paxillin antibodies and phalloidin (Figure 4B). Fln-depleted cells exhibited small foci of paxillin accumulation and a disorganized actin cytoskeleton that was deficient in stress fibers compared with untransfected controls in the same field (Figure 4C). Because these controls were in fact FlnB–/– MEFs, it was important to examine FlnA localization in wild-type cells. We observed FlnA enrichment in several cytoskeletal structures, including transverse arcs surrounding the endoplasm, nodes of cross-linking connecting mature stress fibers extending above the endoplasm to the transverse arc ring below, as well as at the leading edge and the trailing ends of mature focal adhesions (Figure 4A). The smaller adhesions in Fln-depleted MEFs could be a result of less β1 integrin expression; however, this was found not to be the case (Figure 4D).


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 depletion causes adhesion defects. (A) Merged confocal sections of FlnB+/+ MEF 30 min after plating on FN-coated glass. Scale = 20 μm; green = paxillin, red = phalloidin, blue = FlnA). Enlarged regions highlight areas of FlnA enrichment, including large nodes at stress fiber junctions, the cell edge, adhesions, and transverse arcs surrounding the endoplasm. (B) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for paxillin and phalloidin. GFP-expressing cells were considered transfected and therefore Fln-depleted. Scale = 50 μm. (C) Merged images of paxillin and F-actin panels show that Fln-depleted MEFs exhibited a highly disorganized, stress fiber-free, actin meshwork surrounding the endoplasm as well as a lack of focal adhesions compared with nontransfected cells. Scale = 50 μm; green pseudocoloring = paxillin, magenta = phalloidin). (D) FlnB–/– MEFs were transfected with nontargeting or FlnA-targeting shRNA expression vectors, sorted by FACS, and lysed. GFP-expressing cells from these populations exhibit no difference in beta 1 integrin expression. (E) Fln-depleted MEFs and controls expressing paxillin-RFP were spread on FN-coated glass and imaged with TIRF microscopy. Scale = 5 μm. (F) Kymographs taken along white lines in E show that focal adhesions do not retain their stability over the course of spreading in Fln-depleted MEFs. Scale = 5 μm; 3 min. (G) Fln-depleted MEFs and controls were trypsinized, resuspended in 10 μM nocodazole in serum-free DMEM for 30 min, and allowed to spread. Red arrows indicate protrusive regions that collapse in subsequent frames. Scale = 20 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Fln depletion causes adhesion defects. (A) Merged confocal sections of FlnB+/+ MEF 30 min after plating on FN-coated glass. Scale = 20 μm; green = paxillin, red = phalloidin, blue = FlnA). Enlarged regions highlight areas of FlnA enrichment, including large nodes at stress fiber junctions, the cell edge, adhesions, and transverse arcs surrounding the endoplasm. (B) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for paxillin and phalloidin. GFP-expressing cells were considered transfected and therefore Fln-depleted. Scale = 50 μm. (C) Merged images of paxillin and F-actin panels show that Fln-depleted MEFs exhibited a highly disorganized, stress fiber-free, actin meshwork surrounding the endoplasm as well as a lack of focal adhesions compared with nontransfected cells. Scale = 50 μm; green pseudocoloring = paxillin, magenta = phalloidin). (D) FlnB–/– MEFs were transfected with nontargeting or FlnA-targeting shRNA expression vectors, sorted by FACS, and lysed. GFP-expressing cells from these populations exhibit no difference in beta 1 integrin expression. (E) Fln-depleted MEFs and controls expressing paxillin-RFP were spread on FN-coated glass and imaged with TIRF microscopy. Scale = 5 μm. (F) Kymographs taken along white lines in E show that focal adhesions do not retain their stability over the course of spreading in Fln-depleted MEFs. Scale = 5 μm; 3 min. (G) Fln-depleted MEFs and controls were trypsinized, resuspended in 10 μM nocodazole in serum-free DMEM for 30 min, and allowed to spread. Red arrows indicate protrusive regions that collapse in subsequent frames. Scale = 20 μm.
Mentions: MT targeting is believed to be involved in focal adhesion dynamics (Kaverina et al., 1999; Krylyshkina et al., 2002, 2003). Because MTs do not extend to the periphery in Fln-depleted cells, peripheral focal adhesions may also be altered. To test this possibility, Fln-depleted MEFs and untransfected controls were spread for 1 h and stained with anti-paxillin antibodies and phalloidin (Figure 4B). Fln-depleted cells exhibited small foci of paxillin accumulation and a disorganized actin cytoskeleton that was deficient in stress fibers compared with untransfected controls in the same field (Figure 4C). Because these controls were in fact FlnB–/– MEFs, it was important to examine FlnA localization in wild-type cells. We observed FlnA enrichment in several cytoskeletal structures, including transverse arcs surrounding the endoplasm, nodes of cross-linking connecting mature stress fibers extending above the endoplasm to the transverse arc ring below, as well as at the leading edge and the trailing ends of mature focal adhesions (Figure 4A). The smaller adhesions in Fln-depleted MEFs could be a result of less β1 integrin expression; however, this was found not to be the case (Figure 4D).

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