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First published online 12 May 2009
doi: 10.1242/jcs.044602

Journal of Cell Science 122, 1800-1811 (2009)
Published by The Company of Biologists 2009
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Molecular dissection of the ILK-PINCH-parvin triad reveals a fundamental role for the ILK kinase domain in the late stages of focal-adhesion maturation

Fabio Stanchi1,2, Carsten Grashoff2,*, Carine Flore Nguemeni Yonga1,*, Dominique Grall1, Reinhard Fässler2 and Ellen Van Obberghen-Schilling1,{ddagger}

1 Institute of Developmental Biology and Cancer Research, University of Nice-Sophia Antiopolis, CNRS-UMR6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice, France
2 Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany


Figure 1
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  		Fig. 1. Impaired adhesion maturation upon ILK or PINCH1 deletion. (A) ILKfl/fl and ILK–/– cells plated on FN-coated coverslips and stained with antibodies against adhesion components. Scale bar: 20 µm. (B,C) Western blot of protein extracts (B) and RT-PCR on RNA extracts (C) from ILKfl/fl, ILK–/–, PINCH1fl/fl and PINCH1–/– cells. (D) Immunoprecipitation of endogenous ILK in extracts of ILKfl/fl and ILK–/– (negative control) cells.

 

Figure 2
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  		Fig. 2. Altered subcellular localization of tensin after ILK or PINCH1 deletion. (A) Western blot analysis of ILK and PINCH1 cells and clones expressing Flag-RFP-tensin (ILKT and PINCH1T), detected using antibody against Flag. (B) Anti-integrin {alpha}5 and anti-PY staining of ILKT and PINCH1T cells on FN-coated coverslips. Scale bar: 20 µm. (C) Percentage of tensin colocalization with {alpha}5 integrin (±s.d., n=7) determined using the Metamorph colocalization function on images of single cells.

 

Figure 3
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  		Fig. 3. Instability of ILK- and PINCH-null adhesions. (A) Phalloidin, anti-{alpha}-tubulin and anti-{alpha}5 integrin staining of cells plated on FN-coated glass coverslips and treated, or not treated, with 10 µM nocodazole for 45 minutes. Scale bar: 20 µm. (B) Time-lapse analysis of nocodazole and Y27632 treatment of ILK and PINCH1 cells. Cells plated on tissue culture dishes for 8 hours were filmed for 60 minutes before and 90 minutes after the addition of compounds (time 0), with frame rate (time interval between each frame) 1 minute. The percentage of round cells is indicated (mean of three different films, n≥40 cells/film). Extracts from the videos are available (supplementary material Movies 1-4); images from supplementary material Movies 1 and 3 correspond to the indicated times, in minutes (scale bar: 50 µm). (C,D) Time-lapse analysis of the effect of increased cytoskeletal tension on adhesions in ILKTAfl/fl and ILKTA–/– cells, plated on FN-coated coverslips and filmed immediately after the addition of 10 µM nocodazole (frame rate 20 seconds). The first frame on the left corresponds to the time of nocodazole addition and the relative time (`, minutes; `', seconds) is indicated in subsequent frames (C). Arrowheads indicate patches of RFP-tensin moving centripetally with F-actin. Scale bar: 20 µm. Complete videos are available (supplementary material Movies 5 and 6). (D) Anti-{alpha}5 staining on the same cells, fixed immediately after the time lapse.

 

Figure 4
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  		Fig. 4. Interdependency of the IPP components and constitutive targeting of the complex to β3 integrin. (A,B) Western analysis of ILK, PINCH1 and {alpha}-parvin levels in protein extracts from ILK and PINCH1 cells (A), or (B) ILKfl/fl cells stably infected with control or {alpha}-parvin-targeting shRNA lentiviruses (65 or 67). (C-F) Expression of integrin β3-GFP and β3-GFP-IPP chimeras in a clone of 3T3/β3-RFP cells. Western blotting on total lysates (C) and endogenous ILK, PINCH1 and {alpha}-parvin co-immunoprecipitation with the β3-GFP-IPP chimeras using an antibody against GFP (D). (E) Anti-PY staining. Arrowheads denote FAs in which each of the β3-GFP-IPP chimeras is recruited more efficiently than the co-expressed β3-RFP. (F) Differential distribution of each β3-GFP-IPP chimera with respect to the β3-RFP internal control in migrating cells. The black arrow indicates the presumed direction of migration, as deduced by cell morphology. (G) Staining of NIH3T3 cells expressing Flag-RFP-tensin together with β3-GFP (control) or β3-GFP-parvin. Arrowheads mark PY-poor and tensin-rich adhesions in which the β3-GFP-IPP chimera, but not the β3-GFP control, is enriched. Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. Expression of the β3-GFP-IPP chimeras in PINCH1–/– cells rescues defective tensin localization. (A) Western analysis on extracts from PINCH1Tfl/fl, PINCH1T–/– (controls) and PINCH1T–/– cells expressing β3-GFP or one of the three β3-GFP-IPP chimeras. (B-E) Staining with different FA markers: PY (B), {alpha}v (C) and {alpha}5 (D) integrins, and vinculin (E). Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. Dissection of the IPP complex function by expression of deletion mutants of the β3-GFP-IPP chimeras in a clone of 3T3/β3-RFP cells. (A) Detection of the expressed constructs by western blots on total lysates. (B) Co-immunoprecipitation of the chimeric proteins with an antibody against GFP and detection of endogenous IPP complex proteins in the co-precipitate. (C) Differential localization of all β3-GFP-IPP chimeras with respect to β3-RFP. Average i.s.d. values (s.d.) obtained by processing GFP:RFP ratio images of 12 cells from each population, as described in supplementary material Fig. S3. The dashed area indicates the system background noise i.s.d. (D) Combined results of co-immunoprecipitation and fluorescence ratio image analysis. Different protein domains in PINCH1, ILK and {alpha}-parvin proteins and their interactions (bold broken lines) are represented (Ank, ankyrin repeat; Kin, kinase domain). `β' indicates sites of fusion of integrin β3-GFP with each deletion mutant; co-immunoprecipitated proteins of the IPP complex are indicated. The differential localization of constructs (as compared with the internal control β3-RFP) determined in the ratio image analysis is indicated (Y, yes; N, no). The bar below specifies the `region' within the IPP complex endowed with this ability.

 

Figure 7
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  		Fig. 7. Stabilization of IPP components depends on their direct interaction. Western blot analysis on lysates of PINCH1T–/– (A) or ILKT–/– cells (B) expressing the set of β3-GFP-IPP deletion mutants. Schematics below show the interactions between the chimeric proteins and endogenous IPP components that lead to their stabilization in the PINCH1-null and ILK-null backgrounds, respectively.

 

Figure 8
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  		Fig. 8. Adhesion targeting of ILK kinase domain mutants in ILK–/– cells and effects of {alpha}-parvin silencing in ILKT–/– cells expressing β3-GFP-{Delta}Ank. (A) Quantification of spreading as compared with β3-GFP control cells (n≥150; ***P<0.001). (B) Co-immunoprecipitation of the constructs with antibody against GFP and detection of paxillin and {alpha}-parvin by western blotting. (C) Localization of the chimeric constructs and tensin. (D,E) ILKT–/– cells expressing β3-GFP-{Delta}Ank were stably infected with control or {alpha}-parvin-targeting shRNA lentiviruses (65 and 67), then analyzed by western blot (D) and stained as above (E). (F) ILKT–/– cells expressing β3-GFP-parvin plated on coverslips. Scale bars: 20 µm.

 

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© The Company of Biologists Ltd 2009