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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 results in a reduction of ER spread area. (A) FlnB–/– MEFs transfected with negative control shRNA vector and RFP-ER were plated on FN-coated glass for 30 min and fixed. GFP signal served as a marker for shRNA transfection. Scale = 20 μm. (B) Same as in A, but transfected with FlnA-targeting shRNA. (C) ER area was measured by either RFP signal or DIC and normalized to the whole cell area as measured by DIC (***p < 0.001, n = 44). (D and E) Time-lapse images of negative control and Fln-depleted MEFs on FN. Scale = 20 μm. (F) Normalized endoplasm area over time (n = 8, p < 0.001).
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Figure 2: Fln depletion results in a reduction of ER spread area. (A) FlnB–/– MEFs transfected with negative control shRNA vector and RFP-ER were plated on FN-coated glass for 30 min and fixed. GFP signal served as a marker for shRNA transfection. Scale = 20 μm. (B) Same as in A, but transfected with FlnA-targeting shRNA. (C) ER area was measured by either RFP signal or DIC and normalized to the whole cell area as measured by DIC (***p < 0.001, n = 44). (D and E) Time-lapse images of negative control and Fln-depleted MEFs on FN. Scale = 20 μm. (F) Normalized endoplasm area over time (n = 8, p < 0.001).

Mentions: The spreading defect associated with Fln depletion appeared to be affecting the spreading of the endoplasm, a region previously described as the slightly raised region near the cell center containing the majority of membrane-bound organelles and surrounded by the ectoplasm, the peripheral region of dense actin arrays that exclude organelles (Nishizaka et al., 2000). To confirm the molecular nature of this region, we transfected Fln-depleted MEFs and controls with a construct expressing a red fluorescent protein (RFP)-tagged endoplasmic reticulum (ER) marker, namely the calreticulin ER targeting sequence and the KDEL retention sequence. Fixation of transfected cells showed a smaller ER area in Fln-depleted MEFs (Figure 2B) compared with controls (Figure 2A). Because ER area is highly dependent on overall cell area, we measured the ratio of fluorescent ER area to differential interference contrast (DIC) whole cell area to determine the percentage of total area occupied by the ER. Fln-depleted MEFs exhibited an ∼40% decrease in normalized ER area compared with controls (Figure 2C), confirming that Fln depletion causes a spreading defect of the ER. Importantly, we observed no significant difference between the area of Fln-depleted MEFs and controls, whereas the difference in ER areas was significant, even before normalization. Additionally, the vesicle-rich endoplasm was measured using video-enhanced DIC, and the area of the endoplasm was equal to the area of the ER (Figure 2C), showing that using DIC micrographs alone was suitable for analyzing ER area.


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 results in a reduction of ER spread area. (A) FlnB–/– MEFs transfected with negative control shRNA vector and RFP-ER were plated on FN-coated glass for 30 min and fixed. GFP signal served as a marker for shRNA transfection. Scale = 20 μm. (B) Same as in A, but transfected with FlnA-targeting shRNA. (C) ER area was measured by either RFP signal or DIC and normalized to the whole cell area as measured by DIC (***p < 0.001, n = 44). (D and E) Time-lapse images of negative control and Fln-depleted MEFs on FN. Scale = 20 μm. (F) Normalized endoplasm area over time (n = 8, p < 0.001).
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Figure 2: Fln depletion results in a reduction of ER spread area. (A) FlnB–/– MEFs transfected with negative control shRNA vector and RFP-ER were plated on FN-coated glass for 30 min and fixed. GFP signal served as a marker for shRNA transfection. Scale = 20 μm. (B) Same as in A, but transfected with FlnA-targeting shRNA. (C) ER area was measured by either RFP signal or DIC and normalized to the whole cell area as measured by DIC (***p < 0.001, n = 44). (D and E) Time-lapse images of negative control and Fln-depleted MEFs on FN. Scale = 20 μm. (F) Normalized endoplasm area over time (n = 8, p < 0.001).
Mentions: The spreading defect associated with Fln depletion appeared to be affecting the spreading of the endoplasm, a region previously described as the slightly raised region near the cell center containing the majority of membrane-bound organelles and surrounded by the ectoplasm, the peripheral region of dense actin arrays that exclude organelles (Nishizaka et al., 2000). To confirm the molecular nature of this region, we transfected Fln-depleted MEFs and controls with a construct expressing a red fluorescent protein (RFP)-tagged endoplasmic reticulum (ER) marker, namely the calreticulin ER targeting sequence and the KDEL retention sequence. Fixation of transfected cells showed a smaller ER area in Fln-depleted MEFs (Figure 2B) compared with controls (Figure 2A). Because ER area is highly dependent on overall cell area, we measured the ratio of fluorescent ER area to differential interference contrast (DIC) whole cell area to determine the percentage of total area occupied by the ER. Fln-depleted MEFs exhibited an ∼40% decrease in normalized ER area compared with controls (Figure 2C), confirming that Fln depletion causes a spreading defect of the ER. Importantly, we observed no significant difference between the area of Fln-depleted MEFs and controls, whereas the difference in ER areas was significant, even before normalization. Additionally, the vesicle-rich endoplasm was measured using video-enhanced DIC, and the area of the endoplasm was equal to the area of the ER (Figure 2C), showing that using DIC micrographs alone was suitable for analyzing ER area.

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