Consequences of Loss of PINCH2 Expression in Mice

Research Article 5899 Consequences of loss of PINCH2 expression in mice

Fabio Stanchi1, Randi Bordoy1, Oliver Kudlaceck1, Attila Braun1, Alexander Pfeifer2, Markus Moser1 and Reinhard Fässler1,* 1Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany 2Department of Pharmacy, Center for Drug Research, University of Munich, 81377 Munich, Germany *Author for correspondence (e-mail: [email protected])

Accepted 12 September 2005 Journal of Cell Science 118, 5899-5910 Published by The Company of Biologists 2005 doi:10.1242/jcs.02686

Summary PINCH2 belongs, together with PINCH1, to a new family overlapping function in vivo. To further test this possibility, of focal adhesion proteins, the members of which are we established PINCH1-null mouse embryonic fibroblasts, composed of five LIM domains. PINCH1 and PINCH2 which express neither PINCH1 nor PINCH2. We found interact, through their first LIM domain, with the integrin- that in fibroblasts with a PINCH1/2-null background, linked kinase and thereby link integrins with several signal PINCH2 is able to rescue the spreading and adhesion transduction pathways. Despite their high similarity, it has defects of mutant fibroblasts to the same extent as PINCH1. been shown that PINCH1 and PINCH2 could exert distinct Furthermore, we show that the LIM1 domain only of either functions during cell spreading and cell survival. To PINCH1 or PINCH2 can prevent ILK degradation despite investigate the function of PINCH2 in vivo, we deleted their failure to localize to focal adhesions. Altogether these PINCH2 in mouse using the loxP/Cre system. In contrast results suggest that PINCH1 and PINCH2 share to the PINCH1-deficient mice, which die at the peri- overlapping functions and operate dependently and implantation stage, PINCH2-null mice are viable, fertile independently of their subcellular localization. and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder Supplementary material available online at and kidney revealed no apparent abnormalities, but http://jcs.biologists.org/cgi/content/full/118/24/5899/DC1 showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, Key words: PINCH (Lims), ILK, Integrin, Adhesion, Migration

Introduction function in vivo came from studies on UNC-97, the Journal of Cell Science Integrins are heterodimeric transmembrane glycoproteins that Caenorhabditis elegans orthologue of PINCH (Hobert et al., mediate cell-cell and cell-extracellular matrix adhesion 1999). Worms in which UNC-97 has been depleted by RNA (Hynes, 2002). They play pivotal roles in many biological interference arrest their development and show paralysis due processes including cell shape modulation, proliferation, to disrupted muscle attachment sites to the hypodermis. This survival and differentiation (van der Flier and Sonnenberg, phenotype resembles the defects observed in ␤ integrin- and 2001). Integrins execute these functions by recruiting a large ILK-null worms (Gettner et al., 1995; Mackinnon et al., 2002). number of intracellular adaptor and signaling molecules to Similarly, Drosophila melanogaster lacking the PINCH gene their short cytoplasmic domain (Zamir and Geiger, 2001). They suffers from muscle malfunction and paralysis caused by link integrins with different signaling pathways and connect impaired integrin-dependent cytoskeletal attachment to the integrin adhesion sites to the actin cytoskeleton. Integrin- plasma membrane (Clark et al., 2003). associated molecules can also modulate integrin activation Vertebrates have two PINCH isoforms, PINCH1 and (affinity) and their clustering in focal adhesion sites (FAs; PINCH2 (Braun et al., 2003; Zhang et al., 2002c). Both PINCH avidity). One FA protein is PINCH (also known as Lims), proteins share the same modular architecture and have high which is composed of five LIM domains followed by a short sequence similarity. The most noticeable difference between C-terminal tail containing putative nuclear localization/export the two PINCH proteins is the C-terminal tail, which is 11 signals. Each LIM domain is composed of two zinc-finger amino acids longer in PINCH2 than in PINCH1. In addition, motifs, which are able to engage in protein interactions the two PINCH genes share the same structure (Braun et al., (Kadrmas and Beckerle, 2004). The second zinc-finger of the 2003) suggesting that they have probably evolved by gene LIM1 domain of PINCH binds integrin-linked kinase (ILK) (Li duplication of a common ancestor, as have the members of et al., 1999). The LIM4 domain binds the adaptor protein many other vertebrate gene families (Gu et al., 2003). We NCK2, which interacts with several tyrosine kinase receptors previously compared the expression pattern of the two PINCH thereby linking integrins and growth factor signaling (Tu et al., genes during mouse development and in adult mouse tissues 1998). The LIM5 domain interacts with the Ras suppressor 1 (Braun et al., 2003). Interestingly, both in situ and northern blot (RSU1), which links PINCH to JNK signaling (Kadrmas et al., analysis indicated that only PINCH1 is expressed during early 2004; Dougherty et al., 2005). embryonic development, whereas PINCH2 expression is first The first evidence for an essential role of PINCH for integrin detectable in the second half of embryogenesis. Although in 5900 Journal of Cell Science 118 (24) adult tissues the PINCH1 and PINCH2 transcripts are detected selectively in the primitive endoderm of the implanting in almost all organs, the expression of the two isoforms can embryo (Li et al., 2005). diverge within different cell types of the same organ. For Whereas in vivo and in vitro studies provided insight into example, in colon PINCH2 expression is restricted to the the role of PINCH1, little is known about PINCH2. smooth muscle layer, whereas PINCH1 is also expressed in the Overexpression studies in HeLa cells revealed that PINCH2 epithelial layer (Braun et al., 2003). can compete with PINCH1 for ILK binding and hence alter the In order to analyze PINCH1 function in vivo we and others balance of PINCH1-ILK to PINCH2-ILK complexes (Zhang recently reported the phenotype of PINCH1-deficient mice, et al., 2002c). The appropriate balance seems to be essential which die shortly after implantation (Li et al., 2005; Liang et since re-expression of PINCH1 fully restored all the defects in al., 2005). Although loss of ␤1 integrin or ILK also leads to PINCH1 knock down cells, whereas expression of PINCH2 peri-implantation lethality (Fässler and Meyer, 1995; Stephens rescued ILK protein level, but not cell spreading and Akt et al., 1995; Sakai et al., 2003), PINCH1-null embryos survive phosphorylation (Fukuda et al., 2003; Xu et al., 2005). An significantly longer (Li et al., 2005). This prolonged additional functional difference between PINCH1 and development was confirmed in PINCH1-null embryoid bodies PINCH2 is the inability of PINCH2 to interact with the (EBs), which we generated from PINCH1-null ES cells. The PINCH1-binding protein RSU1 (Dougherty et al., 2005). All EB studies revealed that lack of PINCH1 permits these findings led to the proposal that PINCH2 may act as a differentiation of primitive endoderm and epiblast but their regulator of PINCH1 activity (Wu, 2004). adhesion to the basement membrane (BM), polarity, survival To investigate PINCH2 function in vivo, we generated and, surprisingly, cell-cell adhesion are severely compromised PINCH2 (Lims2 – Mouse Genome Informatics) knockout (Li et al., 2005). mice. To our surprise, these mice are viable, fertile and age In addition to our in vivo studies, many in vitro experiments normally. Tissues that normally express high levels of PINCH2 provided insight into the molecular functions of PINCH1. up-regulated PINCH1 expression. Complementation Overexpression of dominant negative PINCH1 protein or experiments with mouse embryonic fibroblasts (MEFs) that siRNA knock downs of PINCH1 in HeLa cells revealed an express neither PINCH1 nor PINCH2 revealed that PINCH2 is essential role in controlling cell spreading, adhesion and able to rescue their spreading and adhesion defects to the same migration (Fukuda et al., 2003; Zhang et al., 2002a). extent as PINCH1. Interestingly, the subcellular localization of Furthermore, these studies revealed that the interaction of either PINCH isoforms is essential to restore spreading and PINCH1 and ILK is required for the localization of a ternary adhesion but is not essential to prevent ILK degradation. complex composed of PINCH1, ILK and ␣-parvin to integrin adhesion sites as well as for the stability of each component of the complex. Depletion of PINCH1 leads to downregulation of Materials and Methods ILK protein and, vice versa, depletion of ILK leads to ES cells culture and generation of PINCH2-null mice downregulation of PINCH1 through proteasome-mediated A 500 bp fragment from a PINCH2 EST clone (GeneBank accession protein degradation (Fukuda et al., 2003). These findings are number AI325875) was used to screen a PAC library. Several clones in agreement with our PINCH1-null EBs, which also showed were identified and used to generate a conditional targeting PINCH2 Journal of Cell Science reduced ILK protein levels (Li et al., 2005). The molecular construct (PINCH2fl). Briefly, the targeting vector consists of a 4 kb left arm followed by a single loxP site, a 1.6 kb genomic fragment mechanism of how these proteins can mutually prevent their containing exon 3 and 4, a neo-tk cassette flanked by two loxP sites degradation is still unclear. and a 5 kb right arm (for more detailed information please contact Evidence from several sources have suggested a role for [email protected]). The construct was electroporated into R1 mammalian PINCH1 in cell survival. The molecular ES cells and clones that underwent homologous recombination were mechanisms underlying PINCH1-mediated cell survival are isolated, transiently transfected with a Cre expression plasmid and complex and involve several signaling pathways. Knock selected in the presence of 1-2Ј-deoxy-2Ј-fluoro-b-D- down of ILK or PINCH1 in HeLa cells leads to reduced arabinofuranosyl-5-iodouracil (FIAU, Moravek Biochemicals Brea, phosphorylation of PKB/Akt at Ser473 (Fukuda et al., 2003), CA ). Clones that lost the neo-tk cassette but not the genomic fragment which can be phosphorylated by ILK (Delcommenne et containing exon 3 and 4 (floxed allele in Fig. 1A) were identified by al., 1998). In addition to the diminished Ser473 Southern blot analyses and injected into blastocysts to generate germline chimeras. phosphorylation, PINCH1 knock down cells also displayed a PINCH2 mutant mice were genotyped by Southern blot analyses decreased phosphorylation of Thr308, which is (Fig. 1B) or PCR (not shown). For PCR analyses, a forward primer phosphorylated by 3-phosphoinositide-dependent kinase 1 (5Ј-CACTCCCAATTCCCCTCCCTGAG-3Ј) was used in (PDK1) (Alessi et al., 1997). How PINCH1 affects PDK1 combination with a first reverse primer (5Ј-AGGGGTCT- activity and consequently Thr308 phosphorylation is unclear. GAGGTCCTGAGAAGG-3Ј) to determine the wild-type and floxed Finally, expression of a constitutively active PKB/Akt protein allele, or with a second reverse primer (5Ј-GGACAGAGGGG- did not rescue these cells from apoptosis, suggesting that GCAAAG ACC-3Ј) to detect the null allele. PINCH1-dependent survival signaling acts either both upstream and downstream or additionally in parallel to Preparation of primary and immortalized MEF cells PKB/Akt (Fukuda et al., 2003). The gene knockout studies in We previously described the generation of PINCH1+/fl embryonic mice also pointed to a prominent role of PINCH1 in cell stem (ES) cells, in which loxP sites flank exon 4 of the PINCH1 gene survival (Li et al., 2005; Liang et al., 2005). Interestingly, (Li et al., 2005). The PINCH1+/fl ES cells were injected into however, these studies also demonstrated that the survival blastocysts to generate germline chimeras and then the mice were function is cell specific and does not operate in mated to obtain a PINCH1fl/fl mouse strain. Mouse embryonic trophectodermal cells, inner cell mass cells and epiblast but fibroblasts (MEFs) were isolated from PINCH1fl/fl mice at E16.5 PINCH2 knockout mice 5901

Fig. 1. (A) Targeting strategy of PINCH2. Partial map of the PINCH2 wild-type and ﬂoxed alleles, and of the knockout allele after Cre recombination. Exons and loxP sites are indicated as rectangles and triangles, respectively. The DNA fragment sizes obtained after EcoRI restriction digest, as well as the probe used for Southern blotting are indicated. (B) Southern blot on DNA samples from offspring of a PINCH2+/– intercross. (C) Northern blot on poly(A)+ RNA extracts from bladder. (D) Non- quantitative RT-PCR performed with primers hybridizing to exons 2 and 5 of the PINCH2 gene, respectively, and template RNA from bladder of PINCH2+/+ and PINCH2–/– littermates (negative image of the agarose gel). To test the efficiency of reverse transcription, the GAPDH cDNA was ampliﬁed and is shown below. (E) Western blot on protein extracts from bladder, kidney and liver of PINCH2+/+ and PINCH2–/– littermates mice.

following standard procedures (Talts et al., 1999). Part of the MEF mix of rabbit anti-GFP antibody and protein A-Sepharose slurry cells were immortalized by retroviral transduction of the SV40 large (Sigma-Aldrich, St Louis, MO; 3 ␮l antibody + 50 ␮l of protein A- T antigen. Immortalized PINCH1fl/fl MEF cells were cloned and Sepharose per sample) in a final volume of 1 ml. After 2 hours at 4°C, subsequently infected with an adenovirus expressing Cre recombinase the Sepharose was pelletted, washed three times with 1 ml IP buffer (4000 units/cell) to obtain PINCH1–/– clones. Primary MEFs were and finally resuspended in 2ϫ SDS-PAGE loading buffer for western directly infected with a retrovirus expressing Cre recombinase. blot analysis. Deletion of the PINCH1 gene was detected by PCR using specific primers described previously (Li et al., 2005). Antibodies All antibodies have been described previously (Li et al., 2005; RNA isolation, northern blot and RT-PCR Sakai et al., 2003) except rabbit anti-GFP ab290 (Abcam Ltd, Journal of Cell Science RNA isolation from cells and mouse tissues and northern blot assays Cambridge, UK) and mouse monoclonal anti-talin (Sigma- were performed as described previously (Braun et al., 2003). Poly(A)+ Aldrich). Anti-tenascin-C was a kind gift from Andreas Faissner RNA was purified using a commercial kit (Oligotext mRNA mini kit, (Department of Molecular Neurobiology, Ruhr University, Qiagen GmbH, Hilden, Germany) and 1 ␮g mRNA was gel separated Bochum, Germany). Corresponding secondary antibodies were and hybridized with a PINCH1 cDNA probe (Braun et al., 2003), a purchased from Jackson Immunoresearch Laboratories Inc. (West PINCH2 cDNA probe generated by PCR amplification using specific Grove, PA), Molecular Probes (Eugene, OR) and BioRad (Hercules, primers (5Ј-TCTCTAGTGTCAGTCTCTAGT-3Ј and 5Ј-CTTCCC- CA). GTCATGGTGGCCGCG-3Ј), and a ILK cDNA probe spanning a 340 bp SmaI-NsiI fragment excised from the ILK cDNA. For RT-PCR, 5 ␮g of total RNA were reverse-transcribed using an oligo(dT)20 primer Histological analysis and Immunocytochemistry and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA), Histological analysis of mouse tissues was performed as described according to the manufacturer’s protocol. The single strand cDNA was previously (Sakai et al., 2003). For immunocytochemistry, cells used as a template for a PCR reaction, using a forward primer were plated for 4 hours on glass slides coated with 10 ␮g/ml hybridizing to exon 2 (5Ј-GATGCCATGTGCCAGCGCTG-3Ј) and a bovine fibronectin (Sigma-Aldrich), then fixed and stained reverse primer hybridizing to exon 5 (5Ј-GAGCAGCTGAAGTG- using a standard protocol (Sakai et al., 2003). F-actin was GTCCGG-3Ј) of the PINCH2 gene. visualized with TRITC-conjugated phalloidin (Sigma-Aldrich). Samples were analyzed using a confocal microscope (Leica, Bensheim, Germany). Preparation of protein lysates for western blotting and co- immunoprecipitation For PKB/Akt phosphorylation assays, cells were grown to 60-70% FACS assay confluency, serum-starved for 4 hours and stimulated with 10% FCS Cells (2ϫ105) were washed twice in ice-cold PBS, resuspended in 100 for the 10 minutes. Cell lysis and western blotting were performed as ␮l of binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM described previously (Sakai et al., 2003). For co-immunoprecipitation CaCl2) and stained with 5 ␮l of Alexa Fluor 488-conjugated annexin (IP) experiments, sub-confluent cells in 150-mm dishes were lysed in V. After 15 minutes 200 ␮l of binding buffer and 1 ␮g/ml propidium 1 ml IP buffer (1% Triton X-100, 50 mM Tris-HCl pH 7.4, 150 mM iodide were added to each sample (Sigma-Aldrich). Positive control NaCl, 10 mM Na4O7P2, 2 mM Na3VO4 pH 10, 100 mM NaF). For cells were cultured for 24 hours in suspension in serum-free medium each IP, 700 ␮g of each protein extract were mixed with 53 ␮l of a in order to induce apoptosis. 5902 Journal of Cell Science 118 (24)

Retroviral constructs tissues analyzed. Finally, these mice were crossed with deletor- EGFP was directionally cloned using the EcoRI and BamHI sites of Cre mice to generate mice harboring one PINCH2 null allele the pCLMFG retroviral vector (Naviaux et al., 1996), generating a (PINCH2+/–). pCLMFG-EGFP vector that was used for the cloning of all further The deletion of exon 3 and 4 causes a reading frame shift constructs. The following primers were used to amplify PINCH1 and within the first LIM domain and should therefore result, if at PINCH2 portions: LIM1/PINCH1 (5Ј-GCGGATCCGGGGCCA- all, in the expression of a short, non-functional N-terminal ATGCCTTGGCCAG-3Ј and 5Ј-CGGGATCCTCAGCAGGGAGCA- Ј Ј PINCH2 polypeptide. Heterozygous PINCH2 mice AAGAG-3 ), LIM1/PINCH2 (5 -GCGGATCCGGGTCTGAGTGTT- (PINCH1+/fl; Cre) appeared normal and did not show any TGGCCGA-3Ј and 5Ј-CGGGATCCTCAGCATGGAGCAAATAG- 3Ј), ⌬LIM1/PINCH1 (5Ј-GCGGATCCGGCCAAATGCTCTTTGC- overt phenotype. To eliminate the Cre allele, these mice were TCC-3Ј and 3Ј-GCGGATCCTATTATTTCCTTCCTAAGGT-5Ј) and crossed with C57Bl/6 mice and the progeny was genotyped for ⌬LIM1/PINCH2 (3Ј-GCGGATCCGGCCAAATGCTATTTGCTCC- Cre. Mice carrying one PINCH2-null allele and lacking the 5Ј and 3Ј-GCGGATCCTATTAGAGTGAGTTGACGTC-5Ј). Full- deletor-Cre transgene (PINCH2+/–) were intercrossed to length PINCH1 and 2 cDNAs (Braun et al., 2003) and their deletion generate PINCH2-deficient (PINCH2–/–) mice (Fig. 1B). constructs were subcloned in the BamHI site in the pCLMFG-EGFP Among 43 viable, 3-week-old offspring from heterozygous vector. VSV-G-pseudotyped retroviral vectors were produced by intercrosses, 9 were wild type, 21 heterozygous and 13 transient transfection of 293T (human embryonic kidney) cells. Viral homozygous. PINCH2-deficient mice were indistinguishable particles were concentrated from cell culture supernatant as from their wild-type or heterozygous littermates, were fertile previously described (Pfeifer et al., 2000) and used for infection of and did not develop any obvious abnormal phenotype. PINCH1-deficient fibroblasts.

Cell adhesion assay Lack of PINCH2 leads to upregulation of PINCH1 Cell adhesion assays were carried out as described previously (Sakai protein level et al., 2003). Northern blot experiments and in situ hybridizations revealed PINCH2 expression in a number of different tissues (Braun et al., 2003). In order to test whether the PINCH2 gene is Cell migration and cell wounding assays inactivated in our mutant mice we performed northern blots Transwell chambers (Corning Incorporated Life Sciences, Acton, and prepared protein extracts from different organs of wild- MA) with an 8 ␮m pore diameter were coated on the lower surfaces ␮ ϫ 4 type and PINCH2 knockout mice. For northern blot, we with 10 g/ml fibronectin (FN; Sigma-Aldrich). Cells (3 10 ) Ј suspended in 0.1 ml DMEM/1% FCS were placed into the chamber designed a specific probe 5 to the site of deletion and and incubated for 5 hours at 37°C. Afterwards cells in the upper overlapping with exon 1. The PINCH2 mRNA showed up as a + surface of the membrane were removed and the cells on the lower band slightly above 18S rRNA in poly(A) RNA samples surface fixed and stained with 20% methanol/0.1% crystal violet. The extracted from wild-type bladder (Fig. 1C). A fainter band of cells from five randomly chosen microscopic fields were counted and slightly shorter mRNA was detected in poly(A)+ RNA samples cell motility was expressed as the number of the cells/mm2 of the from PINCH2–/– mice (Fig. 1C). To further characterize the microscopic fields±s.d. shorter sized band in mutant tissue we performed RT-PCR on Journal of Cell Science To assay cell migration after cell wounding cells were grown to RNA from bladder of PINCH2+/+ and PINCH2–/– mice, using confluence in six-well plates. The monolayers were wounded by a forward primer hybridizing to exon 2 and a reverse primer scratching with a smoothed glass micropipette. After washing, the hybridizing to exon 5. A band consistent with the expected size cells were incubated in DMEM/10% FCS and observed with a Zeiss of 467 bp was amplified from PINCH2+/+ tissue, whereas a Axiovert microscope. To determine cell polarity the Golgi was –/– localized by GM130 staining. Cells in which the Golgi pointed band around 280 bp was amplified from PINCH2 tissue (Fig. towards the scratch within an angle of 120° were scored positive 1D). Cloning and sequencing of the wild-type and mutant (Etienne-Manneville and Hall, 2001). For each time point at least 300 bands confirmed that the size difference was due to the deletion cells were examined. of 188 nucleotides corresponding to exons 3 and 4. In the mutant PINCH2 mRNA, exon 2 was spliced to exon 5, causing a reading frame shift and introducing a premature stop codon Results in exon 5 (see Fig. S1 in supplementary material). The presence Generation of PINCH2 knockout mice of the premature stop codon probably causes degradation of the To disrupt the murine PINCH2 gene we made a conditional PINCH2 mutant mRNA through nonsense-mediated mRNA PINCH2 targeting vector, in which exons 3 and 4 were flanked decay (reviewed by Wagner and Lykke-Andersen, 2002) which by loxP sites and a neo-tk cassette. Upon electroporation and would account for the weak signal observed in northern blot G418 selection 180 ES cell clones were isolated, and 13 clones assay (Fig. 1C). revealed homologous recombination of the targeting construct We recently generated isoform-specific PINCH1 and within the PINCH2 genomic locus. Out of these, two clones PINCH2 antibodies (Li et al., 2005). Western blots, using the were transiently transfected with a Cre recombinase expression PINCH2 antibody, revealed a 36 kDa band from wild-type vector in order to remove the neo-tk cassette (Fig. 1A). bladder, kidney, liver (Fig. 1E) and other tissue extracts (data Southern blot analyses were used to identify these ES cell not shown). Among all tissues tested, bladder showed the clones (termed PINCH2+/fl). Four PINCH2+/fl ES cell clones highest PINCH2 expression. Long exposure of the blot also were injected into blastocysts to generate chimeric founder revealed weaker signals in kidney, followed by liver, heart and males. After germline transmission, PINCH2+/fl mice were diaphragm (data not shown). No signal could be detected in intercrossed to obtain PINCH2fl/fl mice. These mice were knockout tissues even after long exposure, confirming the normal and expressed normal levels of PINCH2 protein in all inactivation of the PINCH2 gene. PINCH2 knockout mice 5903 Since both PINCH proteins stabilize cellular ILK protein levels (Fukuda et al., 2003) we wondered whether deletion of PINCH2 results in lower ILK levels in vivo. As shown in Fig. 1E, ILK levels were unaltered in tissue extracts from bladder, kidney and liver, as well as in other organs (data not shown). However, PINCH1 protein levels were increased in bladder and also moderately in kidney and liver from PINCH2 knockout mice (Fig. 1E). This increase was also observed in other organs expressing PINCH2 (heart and diaphragm, not shown). In order to test whether the upregulation of PINCH1 occurs at the transcriptional or post-transcriptional level, we performed northern blot analyses. As shown in Fig. 1C, PINCH1 mRNA level was unaltered in mRNA extracts prepared from PINCH2 knockout bladder, suggesting that upregulation of PINCH1 protein is the result of a post-transcriptional mechanism and not by increased mRNA transcription.

Deletion of PINCH2 does not affect the morphology of organs Since PINCH2-deﬁcient mice revealed no obvious phenotype, we performed histological analyses of several tissues. Internal organs from PINCH2–/– mice appeared, from gross inspection to be of normal size and morphology. Tissues that normally express PINCH2 such as bladder, kidney, liver (Fig. 2A-C) and colon (not shown), showed no histological abnormalities. In addition, staining sections for tenascin-C, a protein that is overexpressed during tissue regeneration and many pathological conditions such as inﬂammation and neoplasia (for a recent review see Chiquet- Ehrismann and Tucker, 2004) was normal in knockout tissues (data not shown). Next, we tested whether cells normally expressing PINCH2 upregulate PINCH1 protein. In bladder, both PINCH1 and 2 are expressed in the smooth muscle layer. As shown in Fig. 2D Journal of Cell Science PINCH1 protein expression was elevated in the smooth muscle layer of the bladder of PINCH2 knockout mice.

PINCH2 rescues spreading and adhesion defects of PINCH1-null fibroblasts The absence of an abnormal phenotype of PINCH2-null mice and the upregulation of PINCH1 in PINCH2-deficient tissues suggest that PINCH1 may compensate for the lack of PINCH2, which would imply that the two PINCH isoforms have, at least Fig. 2. Histology of tissue section of PINCH2+/+ (left column) and in part, overlapping functions. To further explore such a PINCH2–/– (right column) (A-C) Haematoxylin and Eosin staining of possibility we decided to test to what extent PINCH1 and 2 can bladder (A), liver (B) and kidney (C) sections; sm, smooth muscle; complement each other in vitro. mu, mucosa; ep, epithelium; bd, bile duct; pa, portal area; cv, central PINCH1 is ubiquitously expressed throughout vein; gl, glomerulus. Bars, 100 ␮m. (D,E) Immunofluorescent embryogenesis. For this reason, we could not develop a cell staining of bladder sections using PINCH1 (D) and PINCH2 (E) model to study the consequences of PINCH2 deletion in the antibodies (in red), counterstained with DAPI (blue); sm, smooth absence of PINCH1 in vitro. However, we recently described muscle; mu, mucosa. Bar, 200 ␮m. the development of ES cells carrying a conditional PINCH1- null (floxed) allele (Li et al., 2005). We used them to generate PINCH1fl/fl mice and then used the mice to derive PINCH1 lacked PINCH2 protein (Fig. 3A), consistent with the finding fl/fl mouse embryonic fibroblast (MEF) cell lines. The that the expression of PINCH2 is limited to a few embryonic PINCH1fl/fl cells were immortalized, cloned and infected with organs (Braun et al., 2003). Hence the mutant cells represent a Cre transducing adenovirus to delete PINCH1. Successful an excellent in vitro system that allows discrimination between deletion was confirmed by western blot analysis and PINCH1 and 2 functions. immunofluorescence staining (Fig. 3A; Fig. 4B). The Consistent with previous reports describing PINCH1- PINCH1–/– cell clones and the parental PINCH1fl/fl cells also depleted HeLa cells (Fukuda et al., 2003), the PINCH1-null 5904 Journal of Cell Science 118 (24) resulted in the formation of abundant stress fibers and well- developed FAs, which were indistinguishable from those present in the parental PINCH1fl/fl cells (Fig. 4C,D). Cell adhesion assays revealed that control cells adhered strongly to FN and vitronectin (VN) and weakly to laminin (LN) but PINCH1-deficient cells showed a clear reduction in adhesion to all ECM substrates analyzed (Fig. 6). Like the abnormal spreading and cell morphology, the adhesion defects could also be rescued with either EGFP-PINCH1 or EGFP- PINCH2 (Fig. 6). Finally, cell migration was tested in a scratch assay. However, PINCH1fl/fl and PINCH1-null cells migrated with the same speed and polarized normally (Fig. S2A-C in supplementary material). In addition, we performed cell motility assay using Transwell chambers in which the undersurfaces of the membranes were coated with FN. Also here no significant difference between PINCH1-null and PINCH1fl/fl cells could be detected (Fig. S2D in supplementary material).

Deletion of PINCH1 in primary MEFs neither impairs survival nor phosphorylation of PKB/Akt Fig. 3. PINCH1fl/fl and PINCH1–/– mouse embryonic fibroblasts It has been reported that the two PINCH isoforms differently (MEFs). (A) Western blot showing the absence of PINCH1 protein in affect cell survival and PKB/Akt phosphorylation of HeLa cells the PINCH1–/– MEFs and of the PINCH2 protein in both (Fukuda et al., 2003). To test these parameters we prepared PINCH1fl/fl and PINCH1–/– MEFs. To control the PINCH2 assay, a primary embryonic fibroblasts from 14.5-day-old PINCH1fl/fl lysate from PINCH2+/+ liver was also blotted. (B-E) Spreading of embryos and transduced them with a retrovirus expressing Cre –/– –/– PINCH1fl/fl (B) and PINCH1 (C) cells and of PINCH1 cells recombinase. Five days after Cre transduction most cells began transduced with EGFP-PINCH1 (D) and EGFP-PINCH2 (E), to retract and 9 days after Cre transduction all of them were respectively. Cells were plated on tissue culture dishes for 16 hours smaller, whereas non-infected MEFs were flat and well spread in the presence of serum. Bar, 20 ␮m. (Fig. 7A,B). PCR genotyping of Cre-transduced cells revealed the loss of the floxed PINCH1 gene (Fig. 7C) and western blot analysis and immunofluorescence staining showed the absence MEFs showed a clear spreading defect, both in the presence of the PINCH1 protein (Fig. 7D-F). Consistent with previously Journal of Cell Science and absence of serum and on both plastic and fibronectin- published data (Fukuda et al., 2003; Li et al., 2005), the level coated glass slides (compare Fig. 3B and C). Infection of these of ILK decreased in the PINCH1-null primary MEFs (Fig. 7D), cells with a retroviral vector expressing EGFP-tagged PINCH1 whereas the level of vinculin was unchanged. Staining of focal rescued the spreading defect (Fig. 3D). Similarly, expression adhesions and F-actin revealed thin focal adhesions and actin of EGFP-PINCH2 also led to normal spreading (Fig. 3E). stress fibers accumulating at the cell cortex of PINCH1–/– Mutant cells infected with a control EGFP retrovirus, however, primary MEFs (Fig. 7E,F). did not rescue the reduced cell spreading (data not shown). Apoptosis was examined by staining cells with Alexa Fluor In addition to the spreading defect, PINCH1-null MEFs 488-conjugated annexin V and the non-vital dye propidium showed fewer FAs, both short and elongated as determined by iodide (PI) and determining the extent of apoptotic and necrotic paxillin staining (Fig. 4A,B). Furthermore, phalloidin staining cells by FACS (Vermes et al., 1995). As a positive control for revealed the presence of a few, thin stress fibers, which tended apoptosis primary PINCH1fl/fl MEFs were cultured in to accumulate cortically. To analyze cell spreading at different suspension and in serum-free medium. Cells undergoing time points, we use time-lapse phase contrast microscopy and apoptosis are positive for annexin V but negative for PI (Fig. performed immunostaining of cells fixed at different times 7G). Both adherent PINCH1fl/fl primary MEFs and PINCH1–/– after seeding on fibronectin. These analyses revealed that the primary MEFs contained around 8% apoptotic cells, whereas floxed and PINCH1-null cells had already adhered 5 minutes the number of apoptotic MEFs grown in suspension rose to after plating and had begun to form focal complexes and to 19.05%. The number of dead cells that were positive for both spread 10 minutes after plating (Fig. 5A,B). The extent of annexin V and PI was similar in the PINCH1fl/fl and spreading, however, was severely reduced. Furthermore, the PINCH1–/– primary MEFs (7.43 and 6.49%, respectively), PINCH1-null cells failed to organize their FA and actin stress whereas in the MEFs grown in suspension the number fibers to the same degree as the parental PINCH1fl/fl cells (Fig. increased to 61.08%. This indicates that loss of PINCH1 in 5C,D). primary fibroblasts does not alter their survival. Next we determined whether expression of EGFP-PINCH2 Next we analyzed the phosphorylation level of PKB/Akt in rescued the actin and FA defects of PINCH1-null MEFs to the the PINCH1 primary MEFs. Western blot cell lysates derived same extent as re-expression of EGFP-PINCH1. We found that from PINCH1fl/fl and PINCH1–/– cells growing in serum- expression of either EGFP-PINCH1 or EGFP-PINCH2 supplemented medium revealed similar levels of PINCH2 knockout mice 5905 Journal of Cell Science

Fig. 4. Immunofluorescent staining of PINCH1fl/fl (A) and PINCH1–/– (B) cells and of PINCH1–/– cells expressing EGFP-PINCH1 (C) or EGFP-PINCH2 (D). Cells were seeded on fibronectin-coated glass coverslips in DMEM without FCS for 4 hours. PINCH1 was either detected by PINCH1 antibody (A,B) or via the EGFP fusion protein (C). PINCH2 was visualized as EGFP-PINCH2 (D). Paxillin was stained with a commercial antibody and filamentous actin by TRITC-conjugated phalloidin. Bar, 20 ␮m.

phosphorylated Ser473 (Fig. 7D). We therefore serum induced PINCH1 or PINCH2 in PINCH1-depleted cells (Fukuda et al., PINCH1fl/fl and PINCH1–/– primary MEFs after starvation and 2003). Furthermore, it has been shown that PINCH1 is also tested PKB/Akt phosphorylation. As shown in Fig. 7H, required for localizing ILK to FAs (Zhang et al., 2002a). It is phosphorylation of Ser473 of PKB/Akt was not significantly unclear, however, whether ILK degradation is a result of its changed in PINCH1-null cells, whereas phosphorylation of intracellular mislocalization or if sole binding of PINCH1 or 2 Thr308 was sometimes normal and sometimes slightly to ILK is already sufficient for stabilization. Furthermore, it is reduced. also unclear whether the two PINCH isoforms prevent ILK degradation through the same mechanism. To answer these questions, we built deletion mutants of both PINCH proteins. ILK is stabilized by PINCH1 and PINCH2 independent of Since PINCH1 and 2 bind ILK through their first LIM domains their subcellular localization (Li et al., 1999; Zhang et al., 2002c), we fused EGFP to LIM1 A recent report showed that PINCH1 knockdown cells have derived from PINCH1 and PINCH2 (LIM1/P1, LIM1/P2). lower ILK protein levels because of a proteasome-mediated Furthermore, we created two other EGFP-tagged mutants, in degradation of the ILK protein (Fukuda et al., 2003). The which the LIM1 domain was removed from PINCH1 and degradation of ILK was prevented by the expression of PINCH2 (⌬LIM1/P1, ⌬LIM1/P2). 5906 Journal of Cell Science 118 (24)

Fig. 5. Spreading of PINCH1fl/fl and PINCH1–/– MEFs. The numbers in each frame indicate the time in minutes from the moment of plating. (A,B) Phase contrast images. PINCH1fl/fl (A) and PINCH1–/– (B) MEFs were plated in DMEM on fibronectin-coated tissue culture dishes, incubated at 37°C and 5% CO2, filmed at intervals with a CCD camera. Bar, 40 ␮m. (C,D) Immunofluorescence staining of PINCH1f/fl (C) and PINCH1–/– cells (D). Cells were plated on fibronectin-coated glass, incubated for the time periods indicated and then fixed and stained. Bar, 20 ␮m. Journal of Cell Science

The constructs were expressed in PINCH1-null MEFs by mutants rescued the cell spreading defect caused by PINCH1 retroviral transduction. In contrast to the full-length PINCH1 deletion. Cell lysates from PINCH1–/– cells expressing the full- and 2 proteins (see Fig. 3D,E), none of the PINCH1 and 2 length EGFP-tagged PINCH1 and PINCH2 or EGFP-tagged LIM1/P1, LIM1/P2, ⌬LIM1/P1 and ⌬LIM1/P2 were analyzed by western blot assay. Probing with an anti-GFP antibody showed robust expression of proteins of the expected size corresponding to each construct (Fig. 8A). Similar to the PINCH1–/– primary MEFs and in agreement with previously published data (Fukuda et al., 2003; Li et al., 2005) the level of ILK protein was decreased in PINCH1-null cells (Fig. 8A,B) but not the level of ILK mRNA. The protein levels of other FA components including talin, vinculin and paxillin were unchanged (Fig. 8A). These data confirm a previous report showing that downregulation of ILK depends on a post- transcriptional mechanism that affects ILK but not other FA proteins (Fukuda et al., 2003). The expression of EGFP-PINCH1 or EGFP-PINCH2 in Fig. 6. Adhesion assay with PINCH1fl/fl and PINCH1–/– cells, and with PINCH1–/– cells transduced with EGFP-PINCH1 and EGFP- PINCH1-null cells restored normal ILK levels, whereas PINCH2, respectively. Adhesion was tested on fibronectin (FN), expression of EGFP alone had no effect (Fig. 8A). In addition, vitronectin (VN) and laminin (LN). Bars indicate percentage of total ILK levels were also restored by expressing either LIM1/P1 or number of cells that adhered at saturating concentrations. Error bars LIM1/P2, but not by expressing ⌬LIM1/P1 or ⌬LIM1/P2. In represent ±s.d. (n=3). order to verify the binding of LIM1/P1 as well as LIM1/P2 PINCH2 knockout mice 5907 Journal of Cell Science

Fig. 7. Deletion of the PINCH1 gene in primary MEFs. (A,B) Phase contrast pictures of untreated (A) and of Cre transduced PINCH1fl/fl (B) primary fibroblasts. Bar, 40 ␮m. (C) Genotyping PCR of the untreated (fl/fl) and of Cre transduced PINCH1fl/fl primary fibroblasts (–/–). A water sample (c-) served as a negative PCR control. (D) Western blot on protein extracts of untreated and of Cre transduced PINCH1fl/fl primary fibroblasts cultured in medium containing 10% FCS. (E,F) Immunofluorescent staining of untreated (E) and Cre-treated (F) PINCH1fl/fl primary fibroblasts. Cells were seeded on fibronectin-coated glass coverslips in the absence of FCS for 4 hours and stained with an affinity purified anti-PINCH1 antibody. Focal adhesions and F-actin were visualized with anti-vinculin antibody and with TRITC-conjugated phalloidin, respectively. Bar, 20 ␮m. (G) FACS analysis of untreated (fl/fl), Cre-treated PINCH1fl/fl primary fibroblasts (–/–) stained with Alexa Fluor 488-conjugated annexin V and propidium iodide (PI); a control population of PINCH1fl/fl primary MEFs was cultured in suspension without serum to induce apoptosis (c +). In each diagram, the lower-right sector includes early apoptotic cells that are positives for annexin V but not for PI; the upper-right sector shows the percentage of dead cells that are positive for both annexin V and PI. (H) Phosphorylation of PKB/Akt in the absence of PINCH1. Cells were starved in 0.2% FCS for 4 hours, and then stimulated for 10 minutes with 10% FCS. Phosphorylation of PKB/Akt was determined in lysates from untreated (fl/fl) and Cre-treated (–/–) primary MEFs either before (0) or after (S) serum stimulation.

with ILK, we performed immunoprecipitation of the EGFP- with Fig. 4C,D). Only very faint signals were observed in a tagged constructs and subsequently detected ILK by western few ECM adhesion sites, especially with the ⌬LIM1/P1 and blotting. As shown in Fig. 8C, both LIM1/P1 and LIM1/P2 co- ⌬LIM1/P2 fusion proteins (arrowheads in Fig. 8). In addition, precipitated ILK. The amount of ILK co-precipitated by all cells expressing ⌬LIM1/P1 or ⌬LIM1/P2 had a strong LIM1/P1 and LIM1/P2 was similar to the amount of ILK co- nuclear EGFP signal, which was rarely observed with the precipitated by the full-length EGFP-PINCH1 and EGFP- LIM1/P1 and LIM1/P2 constructs and never with the full- PINCH 2 proteins. No ILK was co-immunoprecipitated with length EGFP-PINCH1 or EGFP-PINCH2. Consistent with the the ⌬LIM1/P1 and ⌬LIM1/P2 constructs or with EGFP alone. ability of LIM1/P1 and LIM1/P2 to stabilize ILK, Immunofluorescent staining revealed that none of the immunofluorescence analysis showed also that PINCH1-null constructs localized efficiently to FAs (compare Fig. 8D-G cells transfected with these constructs had increased ILK 5908 Journal of Cell Science 118 (24) staining in the cytoplasm. This was not the case in PINCH1- be important under stress situation such as wound healing, null cells expressing EGFP only or the ⌬LIM1/P1 and inflammation, etc. However, we show here that the lack of ⌬LIM1/P2 constructs (data not shown). PINCH2 leads to a significant increase of PINCH1 protein level, resulting from a post-transcriptional mechanism and not from changes in gene transcription. It is noteworthy that Discussion upregulation of PINCH1 occurs in the same cell types that Here we describe the consequences of PINCH2 deletion in would normally express high levels of PINCH2. These vivo. PINCH2-null mice are viable, fertile and show no overt observations, together with the high protein sequence abnormal phenotype. Histological analyses of several organs similarity suggest that PINCH1 may replace PINCH2 function did not reveal any morphological abnormality or evidence of in vivo. impaired functionality. Although it is possible that subtle It is unclear whether PINCH2 can substitute for PINCH1 in alterations escaped our detection, these findings suggest that if vivo. PINCH2 is not expressed during early embryonic PINCH2 acts as a regulator of PINCH1 activity, the regulation development (Braun et al., 2003) and hence absent at the peri- seems not to be essential for viability. It is, however, still implantation period when the PINCH1-null mice die (Liang et possible, though not mandatory, that PINCH2 function could al., 2005; Li et al., 2005). However, a tissue-specific deletion Journal of Cell Science

Fig. 8. Expression of deletion mutants of PINCH1 and PINCH2 in PINCH1-null cells. (A) Western blots of cell protein extracts. Lane 1: PINCH1fl/fl cells; lane 2: PINCH1–/– cells; lanes 3-9: PINCH1–/– cells expressing, EGFP (3), EGFP-PINCH1 (4), EGFP-LIM1/P1 (5), EGFP- ⌬LIM1/P1 (6), EGFP-PINCH2 (7), EGFP-LIM1/P2 (8), EGFP-⌬LIM1/P2 (9). (B) Northern blot of parental fl/fl cells (lane 1) and PINCH1–/– cells (lane 2). (C) Detection of ILK co-immunoprecipitated with the EGFP-tagged PINCH constructs using an anti-GFP antibody. Lane 1: lysate of PINCH1fl/fl cells; lane 2: negative control co-immunoprecipitation of PINCH1fl/fl cells expressing EGFP; lanes 3-8: PINCH1–/– cells expressing, EGFP-PINCH1 (3), EGFP-LIM1/P1 (4), EGFP-⌬LIM1/P1 (5), EGFP-PINCH2 (6), EGFP-LIM1/P2 (7), EGFP-⌬LIM1/P2 (8). (D- G) Localization of the EGFP-tagged constructs LIM1/P1 (D), ⌬LIM1/P1 (E), LIM1/P2 (F), ⌬LIM1/P2 (G); FAs were visualized by vinculin staining and arrowheads indicate points of partial co-localization of the constructs with vinculin. Bar, 20 ␮m. PINCH2 knockout mice 5909 of the PINCH1 gene in cardiac muscle cells does not lead to phosphorylation and to prevent apoptosis, which represents a an abnormal phenotype (Liang et al., 2005) suggesting that the potentially important functional difference between the two remaining PINCH2 expressed in cardiac muscle cells PINCH isoforms (Fukuda et al., 2003; Xu et al., 2005). Since substitutes for PINCH1. Since in vitro evidence indicates the PINCH1-null MEFs showed normal cell survival and functional differences between the two PINCH isoforms PKB/Akt phosphorylation we could not use them to test (Fukuda et al., 2003) it will be important to test whether this whether a similar functional divergence also operates in compensatory mechanism holds true for other cells and tissues. fibroblasts. Owing to the current lack of an in vivo model that allowed Our in vitro experiments with PINCH1-null MEFs also us to determine the identical and the different functional provided some insight into the mechanism of how these properties of the two PINCH isoforms we developed an in vitro proteins prevent ILK degradation. As reported for PINCH1- cell system. Our western blot and immunohistochemistry depleted HeLa cells (Fukuda et al., 2003), loss of PINCH1 in analysis of mouse tissues revealed no organ, tissue or cell MEFs led to a post-transcriptional downregulation of the ILK subpopulation in which PINCH2 is the solely expressed protein, which was fully recovered by the expression of PINCH isoform. Even in organs showing highest PINCH2 PINCH1 or PINCH2 (Fig. 7). However, we found that expression (e.g. bladder), PINCH1 is present and even expression of the LIM1 domain of PINCH1 or 2 was equally upregulated after PINCH2 ablation. For these reasons, we sufficient to rescue ILK levels, despite their inability to localize could not use PINCH2-deficient cells to develop an in vitro to FAs. This demonstrated that the stabilization of at least part system that allowed investigation of PINCH2 deletion or the of the ILK cellular pool depends on the mere binding to the potential complementation of PINCH2 by PINCH1. We were LIM1 of PINCH1 or PINCH2. Findings described in a recent able, however, to establish several PINCH1-null MEF cell lines report by Xu et al. (Xu et al., 2005) provide further support to that because of their embryonic origin do not express PINCH2. this notion, and our preliminary experiments in ILK-null We could therefore use these cell lines as a suitable in vitro fibroblasts indicate that ILK can prevent PINCH1 degradation system to compare the roles of the two PINCH isoforms. in a similar manner. In fact, downregulation of PINCH1 in Deletion of PINCH1 in MEF cells led to impaired spreading ILK–/– MEFs can be blocked by expressing ILK mutants that and adhesion on several substrates, with dramatic changes in are unable to localize in FA (our unpublished observations). the morphology of FAs and the actin cytoskeleton. Altogether these observations are consistent with the finding Nevertheless, despite their abnormal F-actin distribution that the interaction of PINCH, ILK and ␣-parvin occurs before PINCH1-null MEFs established a normal polarity and showed their localization into FAs (Zhang et al., 2002b). Furthermore, normal cell wound closure or migration through Transwell they suggest that this complex system of post translation chambers. The normal migration of PINCH1-null fibroblasts is regulation may account not only for the downregulation of ILK somehow surprising, since cells defective in organizing their and PINCH1 observed in PINCH1 and ILK mutant cells, actin cytoskeleton and in adhesion to ECM are expected to respectively (Fukuda et al., 2003; Sakai et al., 2003; Li et al., show altered motility. In this respect it is worth noting that loss 2005), but also for the upregulation of PINCH1 in PINCH2 of Rac1 expression in macrophages also results in defective mice. It is very possible that the deletion of PINCH2 leaves a spreading and reduced adhesion to FN but still normal number of ILK molecules unbound, which then bind to and Journal of Cell Science migration, indicating that an abnormal actin cytoskeleton is not stabilize more PINCH1. always associated with impaired migration (Wells et al., 2004). In summary, our study shows that mouse development and The defective spreading in PINCH1-null MEFs is in line postnatal aging can proceed without overt abnormalities when with previous studies with HeLa cells (Fukuda et al., 2003). the PINCH2 gene is disrupted. Furthermore, PINCH1 and However, unlike the HeLa cell studies we could fully rescue PINCH2 may have more overlapping functions than previous the spreading defect in PINCH1-null MEFs by expressing studies indicated (Fukuda et al., 2003) and hence may PINCH2. Furthermore, complementation of PINCH1-null compensate each other under certain circumstances. However, MEFs with PINCH2 restored normal organization of the actin whereas the PINCH2 function seems to be dispensable in vivo, cytoskeleton and FAs and rescued the adhesion defect. These we cannot rule out that the PINCH2/ILK/parvin complex still data suggest that the two PINCH isoforms can play similar serves specific tasks under stress situations that are unique to functions both in cell spreading and adhesion in our fibroblast PINCH2 and cannot be rescued by the PINCH1/ILK/parvin cell lines. A possible explanation for the discrepancy between complex. Likewise, PINCH1 may have certain functions our data and the previously published findings could be that (possibly for cell survival) that are not compensated by the effects of PINCH2 on spreading may vary among different PINCH2. It is reasonable to assume that a member of a cell types. structurally related protein family such as PINCH2 that is not Loss of PINCH1 in primary MEFs grown in serum or in expressed in the first half of embryogenesis lost functional serum-free medium did not affect cell survival and PKB/Akt properties that are executed by PINCH1 during this important phosphorylation. This result also differs from PINCH1 developmental period. This and many other assumptions can depleted HeLa cells (Fukuda et al., 2003) suggesting that the be now tested by deleting either PINCH1 or PINCH2 or both PINCH/ILK complex also regulates cell survival and PKB/Akt in a spatially or temporally restricted manner in vivo. signaling in a cell type-specific manner. Our previous observation that apoptosis is detectable only in the endodermal –/– We thank Heidi Sebald and Michal Grejszczyk for excellent layer of PINCH1 EBs (Li et al., 2005) supports the idea of technical assistance, Claudia Nicolae, Tatjana Dorn, Ling-Wei Chang a cell type-specific role of PINCH1 for sustaining cell survival. and Hao-Ven Wang for helpful suggestions, Andreas Faissner for the It has been shown that expression of PINCH2 in the PINCH1 tenascin-C antibody, Werner Müller for the Cre-expression plasmid knockdown HeLa cells failed to restore normal PKB/Akt and Ernst Pöschl for the Alexa Fluor 488-conjugated annexinV. R.B. 5910 Journal of Cell Science 118 (24)

is supported by a PhD fellowship from the University of Copenhagen Li, S., Bordoy, R., Stanchi, F., Moser, M., Braun, A., Kudlaceck, O., and O.K. is supported by an Erwin Schrödinger fellowship from the Wewer, U., Yurchenco, P. D. and Fässler, R. (2005). PINCH1 regulates Austrian Science Foundation (FWF). The work was supported by the cell-matrix and cell-cell adhesions, cell polarity and cell survival during the DFG (SFB413), Fonds der Chemischen Industrie and the Max Planck peri-implantation stage. J. Cell Sci. 118, 2913-2921. Society. Liang, X., Zhou, Q., Li, X., Sun, Y., Lu, M., Dalton, N., Ross, J., Jr and Chen, J. (2005). PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes. Mol. Cell. Biol. 25, 3056-3062. References Mackinnon, A. C., Qadota, H., Norman, K. R., Moerman, D. G. and Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Williams, B. D. (2002). C. elegans PAT-4/ILK functions as an adaptor Reese, C. B. and Cohen, P. (1997). Characterization of a 3- protein within integrin adhesion complexes. Curr. Biol. 12, 787-797. phosphoinositide-dependent protein kinase which phosphorylates and Naviaux, R. K., Costanzi, E., Haas, M. and Verma, I. M. (1996). The pCL activates protein kinase B alpha. Curr. Biol. 7, 261-269. vector system: rapid production of helper-free, high-titer, recombinant Braun, A., Bordoy, R., Stanchi, F., Moser, M., Kostka, G., Ehler, E., retroviruses. J. Virol. 70, 5701-5705. Brandau, O. and Fässler, R. (2003). PINCH2 is a new five LIM domain Pfeifer, A., Kessler, T., Silletti, S., Cheresh, D. A. and Verma, I. M. (2000). protein, homologous to PINCH and localized to focal adhesions. Exp. Cell Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic Res. 284, 239-250. fragment of matrix metalloproteinase 2. Proc. Natl. Acad. Sci. USA 97, Chiquet-Ehrismann, R. and Tucker, R. P. (2004). Connective tissues: 12227-12232. signalling by tenascins. Int. J. Biochem. Cell Biol. 36, 1085-1089. Sakai, T., Li, S., Docheva, D., Grashoff, C., Sakai, K., Kostka, G., Braun, Clark, K. A., McGrail, M. and Beckerle, M. C. (2003). Analysis of PINCH A., Pfeifer, A., Yurchenco, P. D. and Fässler, R. (2003). Integrin-linked function in Drosophila demonstrates its requirement in integrin-dependent kinase (ILK) is required for polarizing the epiblast, cell adhesion, and cellular processes. Development 130, 2611-2621. controlling actin accumulation. Genes Dev. 17, 926-940. Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J. and Dedhar, Stephens, L. E., Sutherland, A. E., Klimanskaya, I. V., Andrieux, A., S. (1998). Phosphoinositide-3-OH kinase-dependent regulation of glycogen Meneses, J., Pedersen, R. A. and Damsky, C. H. (1995). Deletion of beta synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. 1 integrins in mice results in inner cell mass failure and peri-implantation Proc. Natl. Acad. Sci. USA 95, 11211-11216. lethality. Genes Dev. 9, 1883-1895. Dougherty, G. W., Chopp, T., Qi, S. M. and Cutler, M. L. (2005). The Ras Talts, J. F., Brakebusch, C. and Fässler, R. (1999). Integrin gene targeting. suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein Methods Mol. Biol. 129, 153-187. PINCH1 and participates in adhesion-related functions. Exp. Cell Res. 306, Tu, Y., Li, F. and Wu, C. (1998). Nck-2, a novel Src homology2/3-containing 168-179. adaptor protein that interacts with the LIM-only protein PINCH and Etienne-Manneville, S. and Hall, A. (2001). Integrin-mediated activation of components of growth factor receptor kinase-signaling pathways. Mol. Biol. Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell Cell 9, 3367-3382. 106, 489-498. van der Flier, A. and Sonnenberg, A. (2001). Function and interactions of Fässler, R. and Meyer, M. (1995). Consequences of lack of beta 1 integrin integrins. Cell Tissue Res. 305, 285-298. gene expression in mice. Genes Dev. 9, 1896-1908. Vermes, I., Haanen, C., Steffens-Nakken, H. and Reutelingsperger, C. Fukuda, T., Chen, K., Shi, X. and Wu, C. (2003). PINCH-1 is an obligate (1995). A novel assay for apoptosis. Flow cytometric detection of partner of integrin linked kinase (ILK) functioning in cell shape modulation, phosphatidylserine expression on early apoptotic cells using fluorescein motility and survival. J. Biol. Chem. 278, 51324-51333. labelled Annexin V. J. Immunol. Methods 184, 39-51. Gettner, S. N., Kenyon, C. and Reichardt, L. F. (1995). Characterization of Wagner, E. and Lykke-Andersen, J. (2002). mRNA surveillance: the perfect beta pat-3 heterodimers, a family of essential integrin receptors in C. persist. J. Cell Sci. 115, 3033-3038. elegans. J. Cell Biol. 129, 1127-1141. Wells, C. M., Walmsley, M., Ooi, S., Tybulewicz, V. and Ridley, A. J. Gu, X., Wang, Y. and Gu, J. (2002). Age distribution of human gene families (2004). Rac1-deficent macrophages exhibit defects in cell spreading and shows significant roles of both large- and small-scale duplications in membrane ruffling but not migration. J. Cell Sci. 117, 1259-1268. Journal of Cell Science vertebrate evolution. Nature 31, 205-208. Wu, C. (2004). The PINCH–ILK–parvin complexes: assembly, functions and Hobert, O., Moerman, D. G., Clark, K. A., Beckerle, M. C. and Ruvkun, regulation. Biochim. Biophys. Acta 1692, 55-62. G. (1999). A conserved LIM protein that affects muscular adherens junction Xu, Z., Fukuda, T., Li, Y., Zha, X., Qin, J. and Wu, C. (2005). Molecular integrity and mechanosensory function in Caenorhabditis elegans. J. Cell dissection of PINCH-1 reveals a mechanism of coupling and uncoupling of Biol. 144, 45-57. cell shape modulation and survival. J. Biol. Chem. 280, 27631-27637. Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Zamir, E. and Geiger, B. (2001). Molecular complexity and dynamics of cell- Cell 110, 673-687. matrix adhesions. J. Cell Sci. 114, 3583-3590. Kadrmas, J. L. and Beckerle, M. C. (2004). The LIM domain: from the Zhang, Y., Guo, L., Chen, K. and Wu, C. (2002a). A critical role of the cytoskeleton to the nucleus. Nat. Rev. Mol. Cell. Biol. 5, 920-931. PINCH-Integrin-linked kinase interaction in the regulation of cell shape Kadrmas, J. L., Smith, M. A., Clark, K. A., Pronovost, S. M., Muster, N., change and migration. J. Biol. Chem. 277, 318–326. Yates, J. R., 3rd and Beckerle, M. C. (2004). The integrin effector PINCH Zhang, Y., Chen, K., Tu, Y., Velyvis, A., Yang, Y., Qin, J. and Wu, C. regulates JNK activity and epithelial migration in concert with Ras (2002b). Assembly of the PINCH-ILK-ILKBP complex precedes and is suppressor 1. J. Cell Biol. 167, 1019-1024. essential for localization of each component to cell-matrix adhesion sites. J. Li, F., Zhang, Y. and Wu, C. (1999). Integrin-linked kinase is localized to Cell Sci. 115, 4777-4786. cell-matrix focal adhesions but not cell-cell adhesion sites and the focal Zhang, Y., Chen, K., Guo, L. and Wu, C. (2002c). Characterization of adhesion localization of integrin-linked kinase is regulated by the PINCH- PINCH-2, a new focal adhesion protein that regulates the PINCH-1-ILK binding ANK repeats. J. Cell Sci. 112, 4589-4599. interaction, cell spreading, and migration. J. Biol. Chem. 277, 38328-38338.