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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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Fln depletion causes an early spreading defect. (A) Western blot of FlnB–/– MEFs transfected with nontargeting or FlnA-targeting shRNA vectors expressing GFP sorted from untransfected cells by FACS. FlnA levels were reduced 73% ± 5% (SEM) compared with the control (n = 3). (B) Cell lines were spread on FN-coated glass for 45 min and imaged. Endoplasmic regions are smaller in Fln-depleted MEFs compared with controls. Dotted red lines indicate endoplasmic regions. Scale = 20 μm. (C) Human FL FlnA-GFP rescued the endoplasmic spreading defect when transfected into Fln-depleted MEFs. Scale = 20 μm. (D) Fln-depleted MEFs (cell on left) exhibited an early spreading defect of the endoplasm when spread on 10 μg/ml FN-coated glass. Scale = 20 μm. The cells were fixed and immunostained for FlnA, resulting in correlation of GFP expression with FlnA knockdown and the endoplasmic spreading defect. (E) Fln-depleted and negative control MEFs were plated on 10 μg/ml FN-coated glass for 40 min and fixed. Cells were verified for GFP expression, treated with FM4-64 at 5 μg/ml in PBS for at least 5 min and imaged. Scale = 10 μm. Confocal z-scan along the x-axis of cells shown exhibits disparate thickness of endoplasmic region between Fln-depleted and control MEFs. Scale = 10 μm on both axes. (F) Quantification of cell heights over three separate experiments, at least 15 cells counted for cell type, ***p < 0.001.
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Figure 1: Fln depletion causes an early spreading defect. (A) Western blot of FlnB–/– MEFs transfected with nontargeting or FlnA-targeting shRNA vectors expressing GFP sorted from untransfected cells by FACS. FlnA levels were reduced 73% ± 5% (SEM) compared with the control (n = 3). (B) Cell lines were spread on FN-coated glass for 45 min and imaged. Endoplasmic regions are smaller in Fln-depleted MEFs compared with controls. Dotted red lines indicate endoplasmic regions. Scale = 20 μm. (C) Human FL FlnA-GFP rescued the endoplasmic spreading defect when transfected into Fln-depleted MEFs. Scale = 20 μm. (D) Fln-depleted MEFs (cell on left) exhibited an early spreading defect of the endoplasm when spread on 10 μg/ml FN-coated glass. Scale = 20 μm. The cells were fixed and immunostained for FlnA, resulting in correlation of GFP expression with FlnA knockdown and the endoplasmic spreading defect. (E) Fln-depleted and negative control MEFs were plated on 10 μg/ml FN-coated glass for 40 min and fixed. Cells were verified for GFP expression, treated with FM4-64 at 5 μg/ml in PBS for at least 5 min and imaged. Scale = 10 μm. Confocal z-scan along the x-axis of cells shown exhibits disparate thickness of endoplasmic region between Fln-depleted and control MEFs. Scale = 10 μm on both axes. (F) Quantification of cell heights over three separate experiments, at least 15 cells counted for cell type, ***p < 0.001.

Mentions: FlnA levels were knocked down by transfecting FlnB–/– MEFs with an expression vector for FlnA-targeting shRNA, green fluorescent protein (GFP), and a puromycin resistance sequence. A nontargeting shRNA was similarly expressed as a negative control. GFP-expressing cells transfected with either vector were sorted from untransfected cells by fluorescence activated cell sorting (FACS) and subsequently lysed for Western blot analysis. Cells expressing the FlnA-targeting sequence showed an average of ∼75% knockdown when compared with controls (Figure 1A). Additionally, representative immunofluorescence of the GFP-expressing cells with an antibody to FlnA showed an ∼80–85% depletion of FlnA levels compared with controls in the same field (Figure 1D).


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 an early spreading defect. (A) Western blot of FlnB–/– MEFs transfected with nontargeting or FlnA-targeting shRNA vectors expressing GFP sorted from untransfected cells by FACS. FlnA levels were reduced 73% ± 5% (SEM) compared with the control (n = 3). (B) Cell lines were spread on FN-coated glass for 45 min and imaged. Endoplasmic regions are smaller in Fln-depleted MEFs compared with controls. Dotted red lines indicate endoplasmic regions. Scale = 20 μm. (C) Human FL FlnA-GFP rescued the endoplasmic spreading defect when transfected into Fln-depleted MEFs. Scale = 20 μm. (D) Fln-depleted MEFs (cell on left) exhibited an early spreading defect of the endoplasm when spread on 10 μg/ml FN-coated glass. Scale = 20 μm. The cells were fixed and immunostained for FlnA, resulting in correlation of GFP expression with FlnA knockdown and the endoplasmic spreading defect. (E) Fln-depleted and negative control MEFs were plated on 10 μg/ml FN-coated glass for 40 min and fixed. Cells were verified for GFP expression, treated with FM4-64 at 5 μg/ml in PBS for at least 5 min and imaged. Scale = 10 μm. Confocal z-scan along the x-axis of cells shown exhibits disparate thickness of endoplasmic region between Fln-depleted and control MEFs. Scale = 10 μm on both axes. (F) Quantification of cell heights over three separate experiments, at least 15 cells counted for cell type, ***p < 0.001.
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Figure 1: Fln depletion causes an early spreading defect. (A) Western blot of FlnB–/– MEFs transfected with nontargeting or FlnA-targeting shRNA vectors expressing GFP sorted from untransfected cells by FACS. FlnA levels were reduced 73% ± 5% (SEM) compared with the control (n = 3). (B) Cell lines were spread on FN-coated glass for 45 min and imaged. Endoplasmic regions are smaller in Fln-depleted MEFs compared with controls. Dotted red lines indicate endoplasmic regions. Scale = 20 μm. (C) Human FL FlnA-GFP rescued the endoplasmic spreading defect when transfected into Fln-depleted MEFs. Scale = 20 μm. (D) Fln-depleted MEFs (cell on left) exhibited an early spreading defect of the endoplasm when spread on 10 μg/ml FN-coated glass. Scale = 20 μm. The cells were fixed and immunostained for FlnA, resulting in correlation of GFP expression with FlnA knockdown and the endoplasmic spreading defect. (E) Fln-depleted and negative control MEFs were plated on 10 μg/ml FN-coated glass for 40 min and fixed. Cells were verified for GFP expression, treated with FM4-64 at 5 μg/ml in PBS for at least 5 min and imaged. Scale = 10 μm. Confocal z-scan along the x-axis of cells shown exhibits disparate thickness of endoplasmic region between Fln-depleted and control MEFs. Scale = 10 μm on both axes. (F) Quantification of cell heights over three separate experiments, at least 15 cells counted for cell type, ***p < 0.001.
Mentions: FlnA levels were knocked down by transfecting FlnB–/– MEFs with an expression vector for FlnA-targeting shRNA, green fluorescent protein (GFP), and a puromycin resistance sequence. A nontargeting shRNA was similarly expressed as a negative control. GFP-expressing cells transfected with either vector were sorted from untransfected cells by fluorescence activated cell sorting (FACS) and subsequently lysed for Western blot analysis. Cells expressing the FlnA-targeting sequence showed an average of ∼75% knockdown when compared with controls (Figure 1A). Additionally, representative immunofluorescence of the GFP-expressing cells with an antibody to FlnA showed an ∼80–85% depletion of FlnA levels compared with controls in the same field (Figure 1D).

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