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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 diminished MT extension in spreading MEFs. (A) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for MTs. GFP-expressing cells were considered transfected and therefore Fln-depleted. Red lines demarcate cell edges. Scale = 50 μm. (B) Enlarged regions from A showing that Fln-depleted MEFs exhibit a larger distance between the MT boundary and the cell edge than do controls. (C) Quantification of distance from MT boundary to cell edge in Fln-depleted MEFs and controls after 15 min of spreading on FN-coated glass measured in TIRF (at least 10 measurements per cell, n = 3) (***p < 0.001). (D) Quantification of distance from MT boundary to cell edge in Fln-depleted and controls after 30 min of spreading on FN-coated glass measured with epifluorescence (at least 10 measurements per cell, n = 33) (***p < 0.001). (E) Fln-depleted MEFs and controls were transfected with 3x-ensconsin-GFP, allowed to spread, and imaged in TIRF. (F) Kymographs taken along white lines from E show a persistently larger distance between the MT boundary and the cell edge in Fln-depleted MEFs compared with controls. (G) Quantification of MT dynamics during the first 30 min of spreading by assessing EB3-RFP growth velocity. Fln-depleted MEFs exhibited slower MT extension than controls (multiple measurements per cell, n = 10 cells) (***p < 0.001), whereas 50 μM blebbistatin treatment rescued this phenotype (multiple measurements per cell, n = 7 cells) (***p < 0.001). (H) Quantification of actin rearward flow velocities by assessing LifeAct-Ruby dynamics from the initiation of spreading. Kymographs of Fln-depleted MEFs showed slower rearward flow than controls (multiple kymographs per cell, n = 6 cells/line) (***p < 0.001). (I) Rearward flow velocities gathered from cells spread for at least 30 min. Fln-depleted MEFs show no significant difference from controls (n = 11 cells/line).
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Figure 3: Fln depletion causes diminished MT extension in spreading MEFs. (A) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for MTs. GFP-expressing cells were considered transfected and therefore Fln-depleted. Red lines demarcate cell edges. Scale = 50 μm. (B) Enlarged regions from A showing that Fln-depleted MEFs exhibit a larger distance between the MT boundary and the cell edge than do controls. (C) Quantification of distance from MT boundary to cell edge in Fln-depleted MEFs and controls after 15 min of spreading on FN-coated glass measured in TIRF (at least 10 measurements per cell, n = 3) (***p < 0.001). (D) Quantification of distance from MT boundary to cell edge in Fln-depleted and controls after 30 min of spreading on FN-coated glass measured with epifluorescence (at least 10 measurements per cell, n = 33) (***p < 0.001). (E) Fln-depleted MEFs and controls were transfected with 3x-ensconsin-GFP, allowed to spread, and imaged in TIRF. (F) Kymographs taken along white lines from E show a persistently larger distance between the MT boundary and the cell edge in Fln-depleted MEFs compared with controls. (G) Quantification of MT dynamics during the first 30 min of spreading by assessing EB3-RFP growth velocity. Fln-depleted MEFs exhibited slower MT extension than controls (multiple measurements per cell, n = 10 cells) (***p < 0.001), whereas 50 μM blebbistatin treatment rescued this phenotype (multiple measurements per cell, n = 7 cells) (***p < 0.001). (H) Quantification of actin rearward flow velocities by assessing LifeAct-Ruby dynamics from the initiation of spreading. Kymographs of Fln-depleted MEFs showed slower rearward flow than controls (multiple kymographs per cell, n = 6 cells/line) (***p < 0.001). (I) Rearward flow velocities gathered from cells spread for at least 30 min. Fln-depleted MEFs show no significant difference from controls (n = 11 cells/line).

Mentions: Because restriction of the ER to the cell center may result from the restriction of MTs (Waterman-Storer and Salmon, 1998) we stained Fln-depleted MEFs with anti-tubulin antibodies after 1 h on human plasma fibronectin (FN)-coated glass (Figure 3A). MTs in Fln-depleted MEFs were confined to the cell center compared with untransfected cells in the same field (Figure 3B). When the distance between the MT boundary and the cell edge was quantified, the MT boundary was twice as far from the edge in Fln-depleted MEFs as in controls in both total internal reflection fluorescence (TIRF) and epifluorescence (Figure 3, C and D). To determine whether MTs were only partially extending or if they were extending then retracting, we tracked MTs using fluorescent ensconsin (3xEMTB-GFP). MTs were constrained and did not extend to the cell edge during early stages of cell spreading (Figure 3, E and F).


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 diminished MT extension in spreading MEFs. (A) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for MTs. GFP-expressing cells were considered transfected and therefore Fln-depleted. Red lines demarcate cell edges. Scale = 50 μm. (B) Enlarged regions from A showing that Fln-depleted MEFs exhibit a larger distance between the MT boundary and the cell edge than do controls. (C) Quantification of distance from MT boundary to cell edge in Fln-depleted MEFs and controls after 15 min of spreading on FN-coated glass measured in TIRF (at least 10 measurements per cell, n = 3) (***p < 0.001). (D) Quantification of distance from MT boundary to cell edge in Fln-depleted and controls after 30 min of spreading on FN-coated glass measured with epifluorescence (at least 10 measurements per cell, n = 33) (***p < 0.001). (E) Fln-depleted MEFs and controls were transfected with 3x-ensconsin-GFP, allowed to spread, and imaged in TIRF. (F) Kymographs taken along white lines from E show a persistently larger distance between the MT boundary and the cell edge in Fln-depleted MEFs compared with controls. (G) Quantification of MT dynamics during the first 30 min of spreading by assessing EB3-RFP growth velocity. Fln-depleted MEFs exhibited slower MT extension than controls (multiple measurements per cell, n = 10 cells) (***p < 0.001), whereas 50 μM blebbistatin treatment rescued this phenotype (multiple measurements per cell, n = 7 cells) (***p < 0.001). (H) Quantification of actin rearward flow velocities by assessing LifeAct-Ruby dynamics from the initiation of spreading. Kymographs of Fln-depleted MEFs showed slower rearward flow than controls (multiple kymographs per cell, n = 6 cells/line) (***p < 0.001). (I) Rearward flow velocities gathered from cells spread for at least 30 min. Fln-depleted MEFs show no significant difference from controls (n = 11 cells/line).
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Related In: Results  -  Collection

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Figure 3: Fln depletion causes diminished MT extension in spreading MEFs. (A) FlnB–/– MEFs treated with FlnA shRNA were spread for 1 h on FN-coated glass, fixed, and stained for MTs. GFP-expressing cells were considered transfected and therefore Fln-depleted. Red lines demarcate cell edges. Scale = 50 μm. (B) Enlarged regions from A showing that Fln-depleted MEFs exhibit a larger distance between the MT boundary and the cell edge than do controls. (C) Quantification of distance from MT boundary to cell edge in Fln-depleted MEFs and controls after 15 min of spreading on FN-coated glass measured in TIRF (at least 10 measurements per cell, n = 3) (***p < 0.001). (D) Quantification of distance from MT boundary to cell edge in Fln-depleted and controls after 30 min of spreading on FN-coated glass measured with epifluorescence (at least 10 measurements per cell, n = 33) (***p < 0.001). (E) Fln-depleted MEFs and controls were transfected with 3x-ensconsin-GFP, allowed to spread, and imaged in TIRF. (F) Kymographs taken along white lines from E show a persistently larger distance between the MT boundary and the cell edge in Fln-depleted MEFs compared with controls. (G) Quantification of MT dynamics during the first 30 min of spreading by assessing EB3-RFP growth velocity. Fln-depleted MEFs exhibited slower MT extension than controls (multiple measurements per cell, n = 10 cells) (***p < 0.001), whereas 50 μM blebbistatin treatment rescued this phenotype (multiple measurements per cell, n = 7 cells) (***p < 0.001). (H) Quantification of actin rearward flow velocities by assessing LifeAct-Ruby dynamics from the initiation of spreading. Kymographs of Fln-depleted MEFs showed slower rearward flow than controls (multiple kymographs per cell, n = 6 cells/line) (***p < 0.001). (I) Rearward flow velocities gathered from cells spread for at least 30 min. Fln-depleted MEFs show no significant difference from controls (n = 11 cells/line).
Mentions: Because restriction of the ER to the cell center may result from the restriction of MTs (Waterman-Storer and Salmon, 1998) we stained Fln-depleted MEFs with anti-tubulin antibodies after 1 h on human plasma fibronectin (FN)-coated glass (Figure 3A). MTs in Fln-depleted MEFs were confined to the cell center compared with untransfected cells in the same field (Figure 3B). When the distance between the MT boundary and the cell edge was quantified, the MT boundary was twice as far from the edge in Fln-depleted MEFs as in controls in both total internal reflection fluorescence (TIRF) and epifluorescence (Figure 3, C and D). To determine whether MTs were only partially extending or if they were extending then retracting, we tracked MTs using fluorescent ensconsin (3xEMTB-GFP). MTs were constrained and did not extend to the cell edge during early stages of cell spreading (Figure 3, E and F).

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