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First published online 26 February 2008
doi: 10.1242/jcs.013722

Journal of Cell Science 121, 854-864 (2008)
Published by The Company of Biologists 2008
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IKAP localizes to membrane ruffles with filamin A and regulates actin cytoskeleton organization and cell migration

Lars Dan Johansen1,*, Tiina Naumanen1,*, Astrid Knudsen1, Nina Westerlund2, Irina Gromova3, Melissa Junttila2, Christina Nielsen1, Trine Bøttzauw1, Aviva Tolkovsky4, Jukka Westermarck2,5, Eleanor T. Coffey2, Marja Jäättelä1 and Tuula Kallunki1,{ddagger}

1 Apoptosis Department and Center for Genotoxic Stress, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
2 Turku Centre for Biotechnology, Åbo Akademi and University of Turku, Turku, Finland
3 Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark
4 Department of Biochemistry, University of Cambridge, Cambridge, UK
5 Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland


Figure 1
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  		Fig. 1. RNAi depletion of IKAP results in decreased plating efficiency, cell adhesion, spreading and migration. (A) Western blot analysis of IKBKAP-shRNA-transduced MEFs (m1 and m2) and human fibroblasts (h1-h3) after antibiotic selection. % IKAP values represent the quantification of IKAP depletion in human fibroblasts (n=2) and MEFs (n=3). (B) RT-PCR analysis of IKBKAP-shRNA-treated MEFs (means ± s.d.; n=3; triplicates; ***P<0.001) and human fibroblasts (means ± s.d.; n=1; triplicates; ***P<0.001) after antibiotic selection. (C) Evaluation of plating efficiency. Cells were inoculated with a density of 1.5x104 cells/ml for MEFs or 2x104 cells/ml for human fibroblasts and analyzed 24 hours later. Calculation of cell numbers (means ± s.d.; n=3; triplicates; **P<0.01). (D) The level of cell death in designated MEFs was investigated by measuring LDH release 24 hours after plating and again 24 hours later after a change of media (means ± s.d.; n=1; triplicates; *P<0.05). (E) The adhesion of MEFs was analyzed by inoculating 5x104 cells/ml in serum-free media on non-supporting adherent-surface 24-well plates (Sarstedt) for 1.5 hours. Cells were washed with PBS and fixed for 10 minutes with crystal violet stain [CV; 0.5% crystal violet (Sigma) containing 25% methanol]. The excess dye was rinsed off, the cells were re-suspended in Na-citrate buffer (0.1 M Na-citrate, 50% ethanol) and the absorbance (OD) at 570 nm was measured (means ± s.d.; n=3; triplicates; **P<0.01). (F) To analyze cell spreading, CV-stained cells were imaged by 20x magnification on an Olympus IX70 inverted microscope with an Olympus C-5050 digital camera. The cell areas were measured using ImageJ software (http://rsb.info.nih.gov/ij/) and divided by the number of cells present (means ± s.d.; n=3; triplicates; **P<0.01). (G) For cell migration study, the cells were seeded at a density of 1x105 cells/ml and allowed to adhere over night. A wound was inflicted by scraping a 200 µl sterile tip across the cell layer. The cells were incubated for 17 hours followed by imaging at 10x magnification on an Olympus IX70 inverted microscope with an Olympus C-5050 digital camera. Average wound area was quantified in the picture using ImageJ software (means ± s.d.; n=3; **P<0.01).

 

Figure 2
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  		Fig. 2. Reintroduction of human IKAP but not FD-IKAP rescues the plating efficiency, cell adhesion, spreading and migration phenotypes of IKAP-depleted MEFs. (A) Western blot analysis of IKAP-depleted MEFs (m1 and m2) with the reintroduction of wild-type IKAP and FD-IKAP. (B) RT-PCR analysis of IKBKAP in MEFs after IKAP depletion by shRNA and reintroduction of wild-type IKAP or FD-IKAP. Left, RT-PCR with oligonucleotides recognizing mouse IKBKAP; right, RT-PCR with oligonucleotides recognizing the 5' end of the human IKBKAP reintroduced to MEFs. (C) Plating efficiencies; (D) adhesion; (E) spreading; and (F) migration properties of the MEFs upon IKAP depletion (m1 and m2) and reintroduction of designated IKAP expression constructs together with corresponding empty vector (vector), scrambled (Scr) and protein expression vector pBabe controls were analyzed as in Fig. 1 (means ± s.d.; n=3; triplicates; **P<0.01; ***P<0.001). (G) Phase-contrast images of the designated wound-healing assays carried out as described in Fig. 1G.

 

Figure 3
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  		Fig. 3. RNAi depletion of IKAP results in migration defects in rat cerebellar granule neurons that can be rescued by full-length but not FD-IKAP. (A) Migration of cerebellar granule neurons transiently transfected with designated IKBKAP (M2) or scr siRNA oligonucleotide and/or IKBKAP plasmids was quantified and normalized to control levels (means ± s.d.; n=4; **P<0.01; ***P<0.001). (B) Viability of neurons expressing designated IKBKAP or scr siRNA and/or IKBKAP plasmids (means ± s.d.; n=4).

 

Figure 4
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  		Fig. 4. Focal adhesion and actin organization is disturbed in IKAP-depleted MEFs. (A) IKAP-depleted (m2) or control (scr) MEFs were stained for paxillin and vinculin at 1.5 hours after plating and for actin at 24 hours. Human IKAP or FD-IKAP was introduced to some of the murine IKAP-depleted cells as indicated. (B) Quantification of the number of cells with organized paxillin staining (means ± s.d.; n=3; **P<0.01). (C) Quantification of the percentage of cells with organized actin cytoskeleton (means ± s.d.; n=3; **P<0.01). (D) Western blot analysis of protein levels in MEFs transduced with designated IKBKAP shRNA constructs (left panel) or HeLa cells transiently transfected with siRNA oligonucleotides (right panel) for 48 hours. (E) The soluble (S; G-actin) and insoluble (I; F-actin) actin content of HeLa cells transiently expressing H1 IKBKAP siRNA or scr control oligonucleotide was assessed by western blotting. Triton X-100 soluble and insoluble proteins were extracted as described in the Materials and Methods, and lysates were analyzed for IKAP and β-actin. The quantification was made by ImageGauge software. The quantification is representative of two independent experiments. (F) Paxillin and beclin 1 protein levels in western blot analysis of FD-patient fibroblasts and fibroblasts from a healthy control individual. Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. IKAP co-purifies with various cytosolic proteins. (A) Western blot analysis of equal amounts of proteins from cytosolic and nuclear fractions of cIKAP-strep-expressing HEK293 cells. Anti-IKAP antibody was used to detect endogenous IKAP and cIKAP-strep. Western blot analysis of p150 and GRP75 were used to control the purity of the nuclear and cytosolic extracts, respectively. (B,C) One-STrEP-tag purification. HEK293 cells transiently transfected with either empty vector or cIKAP-strep were harvested 2 days after transfection and cytosolic extracts were prepared as described in the Materials and Methods. Purification was performed according to the manufacturer's instructions (IBA). Eluates were concentrated and run on 10% SDS-PAGE. (B) Western blot analysis of cIKAP-strep detected by anti-IKAP antibody in different purification steps. IP, input; FT, flow through. (C) Silver staining of the purified proteins. The indicated proteins were identified by MALDI-TOF-MS. DNPK1, DNA-dependent protein kinase; USP9X, ubiquitin-specific processing protease. (D) Association of IKAP with its C-terminus. HEK293 cells were transiently transfected with either empty vector, or with FD-IKAP-His or cIKAP-His constructs. After 48 hours, cytosolic extracts were prepared and immunoprecipitated with anti-His antibody. Immunoprecipitates were run on SDS-PAGE and analyzed for endogenous IKAP and the His-tagged IKAP fragments.

 

Figure 6
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  		Fig. 6. IKAP co-localizes with filamin A into membrane ruffles. (A) RT-PCR of IKBKAP, VIP and filamin A. cDNA was prepared from RNA isolated from rat sympathetic neurons (SCG), MEFs and mouse brain. Water was used as a negative control. (B) IKAP association with filamin A. HEK293 cells were transiently transfected with filamin A together with either an empty vector or His-tagged full-length IKBKAP. After 48 hours, cytosolic extracts were prepared and immunoprecipitated with anti-His antibody. Immunoprecipitates were run on SDS-PAGE and analyzed for IKAP and filamin A. Asterisk indicates the 190-kD fragment of filamin A. (C) Pull-down experiment of in-vitro-translated IKAP and filamin A. Unlabeled His-tagged IKAP fragments, S35-labeled (*) N-terminal filamin A fragments and cIKAP-His were in vitro translated with TNT T7 coupled reticulosate lysate system (Promega). Designated in vitro translations were combined and pulled down with anti-His antibody. Upper panel, autoradiogram of pull downs; lower panel, western blot of the IKAP inputs with anti-His antibody. (D) IKAP and filamin A co-immunostaining in IKAP-depleted (m2) and control (scr) MEFs. Human wild-type IKAP and FD-IKAP were expressed in murine IKAP-depleted MEFs as indicated. Cells were plated on coverslips for 24 hours, fixed and stained. (E) Quantification of the percentage of cells with filamin A staining at the membrane ruffles (means ± s.d.; n=3; **P<0.01). (F) Western blot analysis of IKAP and FD-IKAP expression. The quantification (His versus GAPDH; H/G) is representative of two independent transfections. (G) Immunostaining of cells using three different anti-IKAP antibodies (upper panels and lower leftmost panel) and image of the expression of IKAP-GFP in HeLa cells (lower rightmost panel). Boxed images show close-ups of the areas indicated by arrowheads. (H) Western blot analysis of filamin A expression in IKAP-depleted MEFs. (I) Migration of cerebellar granule neurons transiently transfected with designated filamin A (F1) or scr siRNA oligonucleotide was quantified and normalized to control levels (means ± s.d.; n=4; ***P<0.001). (J) Viability of neurons expressing designated filamin A or scr siRNA (means ± s.d.; n=4). Scale bars: 20 µm.

 

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© The Company of Biologists Ltd 2008