Valvular dystrophy associated filamin A mutations reveal a new role of its first repeats in small-GTPase regulation

Abstract

Filamin A (FlnA) is a ubiquitous actin binding protein which anchors various transmembrane proteins to the cell cytoskeleton and provides a scaffold to many cytoplasmic signaling proteins involved in actin cytoskeleton remodeling in response to mechanical stress and cytokines stimulation. Although the vast majority of FlnA binding partners interact with the carboxy-terminal immunoglobulin like (Igl) repeats of FlnA, little is known on the role of the amino-N-terminal repeats. Here, using cardiac mitral valvular dystrophy associated FlnA-G288R and P637Q mutations located in the N-terminal Igl repeat 1 and 4 respectively as a model, we identified a new role of FlnA N-terminal repeats in small Rho-GTPases regulation. Using FlnA-deficient melanoma and HT1080 cell lines as expression systems we showed that FlnA mutations reduce cell spreading and migration capacities. Furthermore, we defined a signaling network in which FlnA mutations alter the balance between RhoA and Rac1 GTPases activities in favor of RhoA and provided evidences for a role of the Rac1 specific GTPase activating protein FilGAP in this process. Together our work ascribed a new role to the N-terminal repeats of FlnA in Small GTPases regulation and supports a conceptual framework for the role of FlnA mutations in cardiac valve diseases centered around signaling molecules regulating cellular actin cytoskeleton in response to mechanical stress.

Keywords: FilGAP; Filamin A; Mitral valve prolapse; Rac1; RhoA.

Fig. 1

Screening of stable melanoma cell lines expressing FlnA-WT, G288R or P637Q. A. Schematic representation of human Filamin A. The N-terminal actin-binding domain (ABD) is followed by 24 Igl repeats. The Igl repeats are interrupted by two hinges regions (in red). FlnA–G288R and P637Q mutations associated to dystrophy valvular are located on repeats 1 and 4, respectively. B. Western blot analysis of total cell extracts (25 µg) from original M2 (FlnA-deficient), A7, FlnA–WT, G288R and P637Q cell lines. Only clones with a FlnA/actin expression ratio comparable to the original A7 cell line were conserved. C. Selection of FlnA stable cell lines: Immunostaining of the original FlnA–WT expressing cell line A7 (upper left) and those established in the present study FlnA–WT (upper right), G288R and P637Q (lower images) (anti-FlnA antibody, green). Only clones exhibiting over 80% of FlnA positive cells were selected and the arrows indicate the few non-expressing cells in mutant FlnA clones. The nuclei were stained with DAPI (blue) (scale bar: 20 µm).

Fig. 2

Filamin A mutations affect cell morphology and size. A. Differential interference contrast images of M2, A7, FlnA–WT, G288R and P637Q cell lines 6 hours after plating. Note the presence of membrane blebs at the surface of FlnA deficient M2 cells only (arrows). A7 and FlnA–WT cells exhibit large lamellipodia structures with membrane ruffles (arrow heads). Scale bar is 10 µm B. Cells expressing FlnA–G288R and P637Q are smaller than FlnA–WT cells. Histograms indicate the area of the cells (µm²) grown for 6 hrs (left-hand) or 24 hrs (middle). At least 100 cells were measured in 3 experiments. Right-hand histogram indicates the number of cells exhibiting lamellipodia 6 hrs after plating. Error bars show SEM, ***P < 0.001 versus FlnA–WT cells. C. Co-localization of FlnA (green) and actin (phalloidin labeling in red) in FlnA–WT, G288R and P637Q cell lines. Note FlnA immunostaining decorates cell plasma membrane. Co-localization of actin and FlnA appears in yellow in the merged images (scale bar 10 µm).

Fig. 3

Filamin A mutations also affect morphology and size of HT1080 cells. A. Western blots of shRNA knockdown FlnA HT1080 cells (first lane) and transfected with shRNA-resistant FlnA–WT, G288R and P637Q. The blot probed with anti-FlnA shows FlnA expression is restored. B. Immuno-localization of GFP-tagged FlnAs using anti-GFP antibody (green) and actin (phalloidin in red, scale bar 10 µm). Cells transfected with mutant FlnA–G288R and P637Q were smaller and exhibited less lamellipodia (left and right-hand histograms, respectively). ***P < 0.001 versus FlnA–WT transfected cells (n = 170 cells).

Fig. 4

Filamin A mutations impede cell adhesion and migration. A. Percentage of adherent cells remaining on coverslips coated with fibronectin after two washes. Nuclei were stained with DAPI and counted on 10 fields, n = 4, error bars show SEM, **P < 0.01 versus FlnA-KO cells. B. Percentage of cells seeded in the upper chamber of “transwell” filters that migrated overnight in the lower chamber. The cells that had migrated were counted on 6 fields from three experiments n = 3, error bars show SEM, **P < 0.01 versus FlnA-KO (M2) cells. C. Typical images of a wound made at the beginning (t₀) and 24 hrs later (t_{24 h}). Wound healing closure was calculated as the surface of the wound covered by the cells at t_{24 h} and is expressed in % in the histogram, n = 3, error bars show SEM, ***P < 0.001 versus FlnA-WT cells.

Fig. 5

Rac1 and RhoA GTPases activities are modifiedin FlnA–P637Q cells. A.Representative experiment of active Rac1 pull down using GST–Crib (left-hand images). Lower Rac1 activity in FlnA–P637Q cells was quantified in five experiments. The mean data are shown on the graph, n = 4, Error bars show SEM, *P < 0.05. B. Increased RhoA activity was detected in FlnA–P637Q cells (left-hand images). Mean data from five experiments is shown on the graph, Error bars show SEM, **P < 0.01 versus FlnA–WT cells.

Fig. 6

Rac1 and RhoA activities determine FlnA–P637Q and G288R cells spreading. A. FlnA–WT and P637Q cell indexes (CI) measured using xCELLigence system in the presence and absence of the Rac1 inhibitor NSC23766 (10 µM) inFlnA–P637Q (left panel) and the ROCK inhibitor Y27632 (10 µM). (right panel). FlnA–WT data are included in both panels for comparison. Results are means ± SEM of four independent experiments. ** indicates significant difference between FlnA–WT with and without inhibitors calculated by two-way analysis of variance, Bonferroni corrected testing (n = 4, $$$P < 0.001 and **P < 0.01). B. Phase contrast images of FlnA–WT and FlnA–P637Q cells seeded on fibronectin coverslip (10 µg/ml) for 2 hours with or without Y27632 (10 µM). The inhibitor restores lamellipodia like structure in the mutant cells. Scale bar: 20 µm. C. FlnA–WT and G288R cell indexes (CI) measured using xCELLigence system in the presence and absence of the Rac1 inhibitor NSC23766 (10 µM)inFlnA–P637Q (left panel) and the ROCK inhibitor Y27632 (10 µM) (right panel). FlnA–WT data are included in both panels for comparison. Results are means ± SEM of four independent experiments. ** indicates significant difference between FlnA–WT with and without inhibitor calculated by two-way analysis of variance, Bonferroni corrected testing (n = 4, $$$P < 0.001 and **P < 0.01).

Fig. 7

FilGAP is “activated” and involved in the reduced spreading of FlnA-P637Qand G288R cells. A. Example of FilGAP extinction in FlnA-P637Q cells treated with FilGAP SiRNA The blot was probed with anti FilGAP and GAPDH (to control loading, 25 µg). B. Cell index of FlnA-P637Qand G288R cells treated with control and FilGAP SiRNA (left and right panels respectively). FlnA-WT data are included in both panels for comparison. Results are means ± SEM of four independent experiments (n = 4 Error bars show SEM, **P < 0.01.) C. Rac1–GAP activity pull down assay. FlnA-WT, G288R and P637Qmelanoma cells were transfected with FilGAP-HA (input lower blot) and “active” FilGAP pulled down using GST-Rac1-Q61L fusion protein (upper blot). “Active” FilGAP pulled down in mutant FlnA cells more than doubled with respect to FlnA-WT cells. **P < 0.01 versus FlnA-WT cells (n = 4).