Spectrine et adhérence cellulaire (implication de la spectrine dans la formation et les fonctions des podosomes)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

αII-spectrin regulates invadosome stability and extracellular matrix degradation.

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation

αII-Spectrin is localized in different invadosomes.

<p>(A) αII-Spectrin localization in PMA-treated HMEC-1 cells and SrcY527F-MEF cells. Starved HMEC-1 cells were treated for 1hr with PMA (50 ng/ml) in order to induce characteristic invadosome rings (zoom red square). Then, endogenous cortactin and αII-spectrin were stained and αII-spectrin relocalization after invadosome induction visualized. The presence of endogenous αII-spectrin was also seen in invadosome rings from SrcY527-MEF cells. (B) 3D localization of αII-spectrin in invadosomes. Confocal Z-stacks were obtained in SrcY527F-MEF cells stained for cortactin or F-actin. αII-Spectrin dashed lines represent the projected areas (Y section is projected on the right and X section on the bottom of the image), showing preferential αII-spectrin localization at the basis of the invadosome ring. Scale bar: 5μm.</p

αII-Spectrin silencing revealed its role in invadosome formation.

<p>SrcY527F-MEF and HMEC-1 cells were transfected for 96 hr with different shRNA directed against αII-spectrin (Sp-shRNA) or an irrelevant shRNA (Nr-shRNA). (A and B) Western Blot analysis revealed a 60% decrease in αII-spectrin expression. Lamin A/C was used to control protein loading. Results revealed efficiency of shRNA3h and 4h (for HMEC-1), 1m and 3m (for SrcY527F-MEF) on αII-spectrin expression. (C) Invadosomes were counted 96 hr after cell transfection with shRNA (GFP positive cells): 300 transfected cells were counted from at least 3 independent experiments. The percentage of cells forming invadosomes was evaluated in control cells (Nr-shRNA) versus spectrin-depleted cells (Sp-shRNA). αII-Spectrin silencing significantly reduced the percentage of HMEC-1 and SrcY527F-MEF cells forming invadosomes.</p

αII-Spectrin silencing changes invadosome morphologies.

<p>SrcY527-MEF cells were transfected for 72 hr with shRNA αII-spectrin (Sp-shRNA) or irrelevant shRNA (Nr-shRNA). (A) F-actin of transfected cells was stained with phalloidin toxin (red) allowing to visualize invadosome rings. Analysis of cells (10 fields per condition) revealed an increased number of fragmented invadosome rings in spectrin-depleted cells. (B) Invadosome ring diameters and (C) invadosome ring thickness were measured in transfected cells. Around 100 rosettes (corresponding to 20 cells, chosen in random field) were analyzed per conditions in 3 different experiments. Knockdown of αII-spectrin induces a significant decrease in ring diameters due in particular to an increase in the percentage of rings with a small size and a decrease number of large rings. In another side αII-spectrin depletion does not modify rings thickness. Scale bar: 20 μm.</p

GFP αII-spectrin dynamics in invadosomes.

<p>(A) Extracted images from time series (min) from representative observations by TIRF microscopy of living SrcY527F-MEF cells expressing full length of αII-spectrin fused to GFP an invadosome marker fused to RFP, cortactin, or the actin marker Ruby-LifeAct. Ten observation fields were analyzed from at least three different experiments. GFP-αII-spectrin accumulated in isolated or invadosome rings, during their expansion or disorganization. These data link αII-spectrin with the intense actin remodeling associated with invadosome dynamics. (B) To confirm this point, net flux of GFP- αII-spectrin was analyzed by FRAP technology. After a 2.9 sec photobleaching in the red square, αII-spectrin fluorescence starts to reappear after only 3.0 sec, and total recovery of the fluorescence in the photobleached area occurred at 15.0 sec. The invadosome ring is identified by a white dash line here in a portion of cell in the first time. Relevance of the results were obtained by analyzing 10 observation fields from at three different experiments. Scale bar: 3 μm.</p

Knockdown of αII-spectrin decreases cell invasion and matrix degradation activity of invadosomes.

<p>(A) SrcY527F-MEF were transfected for 72 hr with shRNAs (Nr-shRNA and Sp-shRNA) and seeded on Boyden’s chamber coated with a layer of Matrigel in order to obtain an invasion chamber. Around 15 random fields were analyzed per condition and results are presented as pool of three different experiments. After 20 hr, the number of invasive cells was significantly reduced by 35% when spectrin was silenced (Nr-shRNA 18,40 vs. Nr-shRNA 11,85 p<0,01). (B) This decrease of the invasive properties of SrcY527F-MEF depleted for αII-spectrin was associated with a large decrease in the percentage of cells able to digest an extracellular matrix component such as FITC-gelatin (Nr-shRNA 81.53 ± 2.31% vs. Sp-shRNA 19.67 ± 6.17%; p<0,001). (C) More precisely, the percentage of invadosome rings associated with a degradation area was significantly reduced in spectrin-depleted cells (Nr-shRNA 63.25 ± 6.70% vs. Sp-shRNA 28.15 ± 3.00%; p<0.01). (D) Extracted images from time series (min) from representative observations of living SrcY527F-MEFcells transfected for 72 hr with shRNAs (Nr-shRNA and Sp-shRNA) and Ruby-LifeAct. Spectrin depletion impairs matrix degradation as shown by the lack of black areas (Sp-shRNA, 120 min) in the red circles, indicating invadosomes at time 0. Scale bar: 20 μm.</p

Deficiency of αII-spectrin increases the mobile fraction of β3-integrin in invadosome rings.

<p>SrcY527F_MEF cells were transfected for 72 hr with irrelevant shRNA (Nr-shRNA) or shRNA targeting αII-spectrin (Sp-shRNA) along with RFP-actin, paxillin-RFP, RFP-cortactin or β3-integrin RFP plasmids in order to quantify the mobility of these invadosome components (by FRAP) in presence or absence of αII-spectrin. (A) A simple exponential equation model was used to quantify K2 (in sec^-1), which is the inverse of the characteristic time of fluorescence recovery (tau), in order to evaluate the diffusion speed of these invadosome proteins. No significant changes were observed, indicating that αII-spectrin does not affect the net rate of entry of these components in invadosomes. (B) Quantification of the immobile fraction of each of these invadosome components. Immobile fraction of actin, paxillin and cortactin remained constant between control and spectrin-depleted cells. However, αII-spectrin depletion significantly reduced the immobile fraction of β3-integrin in invadosomes. (C) Representative FRAP recovery curves of β3-integrin-RFP in presence (Nr-shRNA) or absence (Sp-shRNA) of αII-spectrin. In absence of αII-spectrin, the number of immobile β3-integrin molecules is more important while the plateau is smaller than in presence of αII-spectrin. Results for quantification were obtained from 3 independent experiments where 10 to 11 cells were analyzed for each experiment.</p