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Mar 22, 2012 - Olivier Debeir4, Christine Decaestecker4, Boris Hinz5, An Staes1,2, Evy ...... Didry, D., M.F. Carlier, and D. Pantaloni, Synergy between actin ...
MCP Papers in Press. Published on March 22, 2012 as Manuscript M111.015099

Cells lacking β-actin are genetically reprogrammed and maintain conditional migratory capacity

Davina Tondeleir1,2,6, Anja Lambrechts1,2,6, Matthias Müller3, Veronique Jonckheere1,2, Thierry Doll3, Drieke Vandamme1,2, Karima Bakkali1,2, Davy Waterschoot1,2, Marianne Lemaistre3, Olivier Debeir4, Christine Decaestecker4, Boris Hinz5, An Staes1,2, Evy Timmerman1,2, Niklaas Colaert1,2, Kris Gevaert1,2, Joël Vandekerckhove1,2, Christophe Ampe1,2.

1

Department of Medical Protein Research, VIB, Ghent, Belgium.

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Department of Biochemistry, UGent, Ghent, Belgium.

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Novartis Institute of Biomedical Research, CH-4002 Basel, Switzerland.

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Laboratory of Image Synthesis and Analysis, Faculty of Applied Sciences, Université Libre de Bruxelles, B-1050

Brussels, Belgium. 5

Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Fitzgerald Building, 150 College Street,

University of Toronto, ON M5S 3E2 Canada. 6

equal contribution

Corresponding author: Prof. Dr. Christophe Ampe, Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, 9000 Gent, Belgium. ; Tel: +32-9-264.9332, Fax : +32-9-264.9488.

Running title: β-actin primarily functions in cell homeostasis

1 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Abbreviations 2D: two dimensional α-SMA: α smooth muscle actin COFRADIC: combined fractional diagonal chromatography EGFP: enhanced gree fluorescent protein ES: embryonic stem cell γ-SMA: γ smooth muscle actin GAPDH: glyceraldehyde phosphate dehydrogenase HET: heterozygous KI: knock in KO: knock out L/H: light over heavy isotope ratio mAb: monoclonal antibody MEF: mouse embryonic fibroblast NA: numeric aperture pAb: polyclonal antibody PBSMT: phosphate buffered saline with methanol ROCK: Rho kinase SEM: standard error of the mean shRNA: short hairpin RNA TGFβ: transforming growth factor β WB: Western blot WT: wild type

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Summary Vertebrate non-muscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Due to presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.

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Introduction Vertebrates express six highly conserved actin isoforms [1] in complex developmental and tissue-specific patterns [2]. The major actin isoforms expressed in non-muscle cells are βand γ-cytoplasmic actin (further referred to as β- and γ-actin). Remarkably, in warm blooded vertebrates these isoforms differ only in four amino acids at the N-terminus [1]. The conserved nature of these substitutions can be interpreted in a scenario in which these isoforms perform redundant functions. Yet, spatial and temporal segregation of these isoforms in the cytoplasm has been observed [3], suggesting specific roles. γ-actin displays a more ubiquitous distribution, whereas β-actin is preferentially located at the leading edge of newly formed cellular compartments and protrusions [4-8]. Given this preferred localization and its ubiquitous expression it is generally accepted that β-actin specifically functions in generating cell protrusion. Consistent with this view is that overexpression of β-actin increases cell speed by increasing areas of protrusion and retraction [4, 9-11]. It is however unclear if other actin isoforms are equally capable of generating cell protrusion and productive cell migration. More recently the presence of actin in the nucleus was recognized, and a role for actin in modulating transcription is increasingly appreciated (reviewed in [12, 13, 14]). Antibodies against β-actin block transcription [15] and nuclear translocation of β-actin is involved in macrophage differentiation [16]. Whereas this univocally demonstrates that β-actin is involved in controlling gene transcription, it is unclear to what extent this occurs and if other actin isoforms can compensate for this nuclear function. Genetic evidence suggests that β-actin is an essential gene. Three knock-out models are available [17-19] and in all cases whole-body knock-out results in embryonic lethality, albeit the stage where it happens is different (in one model after E8.5, [18] and in the two other ones at

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E10.5 [17, 19]). We exploited β-actin-/- (knock-out, KO) mouse model from [17, 19] to create a unique model system: mouse embryonic fibroblasts (MEFs) devoid of β-actin. This enabled us to address long term effects of lack of β-actin function on cellular properties. In line with [18] we find compensatory expression of other actin isoforms. This compensation allows cells to adhere to substrates, to make protrusions and, when ROCK is inhibited, even to migrate which indicates that other actin isoforms can act redundantly for the cytoplasmic functions of β-actin; i.e. providing force for cell protrusion. Increased expression of other actin isoforms, upon loss of βactin, is accompanied by a larger change in the genetic program as evidenced by a differential proteome study. Pathway analysis suggested augmented contractility and TGFβ activation. This changed program, resulting from β-actin deletion, occurs despite the presence of other actins in the nucleus suggesting that the nuclear function of β-actin is more unique.

Experimental procedures Mouse embryonic fibroblasts (MEFs) Creation of the heterozygous β-actin KO mice has been described [17]. The β-actin KI mice were created by recombinase mediated cassette exchange with a pCEHyg-H-ACTb insertion plasmid containing the human β-actin cDNA (see Supplemental Fig. S1B). MEFs were derived from littermates in case of wild type (WT)10, KO1, KO2 and heterozygous (HET)8 cells. MEFs were prepared from individual 10.5-day-old embryos. Head and organs enriched in blood vessels were removed and tissue was minced and dissociated in 25 ml ice-cold 0.25% trypsin solution for about 12 h. The solution was warmed to 37°C and then shaken for a few seconds. After carefully washing, the MEFs were propagated in Dulbecco’s modified Eagle medium with high glucose, 10% fetal bovine serum, 870 mg/l glutamine, 0.1 mM beta-mercaptoethanol and 100 5

units/ml penicillin and 100 µg/ml streptomycin. The cells were immortalized with the pSV51 plasmid expressing SV40 largeT-antigen [20] (http://www.belspo.be/bccm/lmbp.html; accession number LMBP1829) at passage 2. The same protocol was used to prepare fibroblasts from homozygous β-actin KI embryos. Embryonic stem (ES) cells were prepared from embryos at the morula stage from crosses of β-actin EGFP with β-actin Plap mice [17]. To reduce β-actin expression we used shRNAmir V2MM-75091 (Open Biosystems) against the 3’UTR of β-actin encoding a short hairpin inserted in the retroviral vector pSM2c. RHS1704 was used as nonsilencing control. 5 µg endotoxin free plasmids were nucleofected (Amaxa kit MEF2) into 2x106 cells and selection with 0.4 µg/ml puromycin was maintained during 11 days. Two human βactin constructs, one containing the coding sequence with only the ZIP-code [21] and with the full-length 3’UTR, were cloned in pMSCV-puro (Clontech). Virus was produced in Human Embryonic Kidney HEK293 cells and KO1 MEFs were transduced and selected with 0.4 µg/ml puromycine during 10 days. Cells were either used for WB or for migration experiments. Antibodies Antibodies used in this study are pan-actin mAb (clone C4), β-actin mAb (clone AC-15), αsmooth muscle actin (α-SMA) mAb (clone 1A4), vinculin mAb (hVIN1), tropomyosin mAb (clone TM311), ADF pAb (clone GV-13), α-tubulin mAb (clone T6199) and B23 mAb (clone FC82291) (all from Sigma), γ-actin pAb (Chemicon and generous gift from J. Ervasti, Department of Biochemistry, University of Minnesota), γ-smooth muscle actin (γ-SMA) mAb, phospho-LIM kinase 1 and 2 pAb (Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pAb (US Biological), MLC2 pAb, phospho-Ser19 MLC2 mAb, MYPT1 pAb and phospho-Thr853-MYPT1 pAb, β-tubulin mAb (9F3) and cofilin, phospho-cofilin, LIM-kinase 1

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and 2 (all from Cell Signaling), Filamin-1 pAb (Santa Cruz), SM22 pAb and calponin pAb (generous gift from M. Gimona, Cytoskeleton Group, University of Salzburg) and LPP pAb (ImmunoGlobe). Secondary Antibodies for WB were goat anti mouse, anti rabbit or anti sheep antibodies coupled to IRDye 800 or IRDye 680 (Li-Cor). Immunohistochemistry Immunohistochemistry on whole mount embryos was performed as previously described [22]. Briefly, embryos were fixed in methanol:DMSO (4:1) overnight at 4°C, treated with methanol:DMSO:H2O2 (4:1:1) for 5-10 hours at room temperature to block endogenous peroxidase activity and stored in methanol at -20°C. The embryos were subsequently rehydrated in 50% methanol in phosphate buffered saline PBS (PBSMT) and incubated with the primary antibodies in PBSMT overnight at 4˚C. Following washes in PBSMT for 5 hours at room temperature, embryos were incubated with an anti-mouse peroxidase-labeled secondary antibody (1/500, NIF825, Amersham Bioscience) in PBSMT overnight at 4°C. Following washes in PBSMT for 5 hours at room temperature and brief washes with PBS with 0.1% Triton X-100, the embryos were developed with 3.3-diaminobenzidine tetrahydrochloride (Vector laboratories). The reaction was stopped by fixing the embryos in 4% paraformaldehyde in PBS at room temperature for 1 hour. Gel electrophoresis and Western blot Total cell or embryonic lysates were prepared in 7 M urea, 2 M thio-urea, 0.5% TritonX-100, 40 mM dithiothreitol and protease inhibitors (1 µg/ml Leupeptin, 1 µg/ml Antipain, 1 µg/ml Aprotinin, 1.6 µg/ml Benzamidine). The amount of loaded protein was 6 µg for one-dimensional and 30 µg for two-dimensional (2D) polyacrylamide gels. For 2D gel electrophoresis the samples were first separated on Immobiline Drystrip gels (pH range 4-7, Amersham Biosciences) and

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subsequently separated according to their molecular weight in 10% polyacrylamide gels. The actin isoform expression patterns or the proteins indicated in the figures were analysed by western blotting (WB) using the appropriate set of primary and secondary antibodies. The protein bands/spots were quantified on an Odysseus scanner. ROCK inhibitor (Calbiochem, Merck KGaA, 10 µM) treated cells were lysed in the same buffer as above, except that 50 µM of ROCK inhibitor was added as well as 1 mM Na-orthovanadaat and 10 mM of NaF. Blots were probed with the antibodies indicated in the figures. Nuclear and cytoplasmic fractions were prepared using the nuclear extract kit from Active Motif. qRT-PCR Target specific primers (see Supplemental Table 4) were designed using Lightcycler Probe Design Software 2.0 (Roche) and synthesized by Proligo (Sigma). Primer sets were validated in silico by N-blasting against the mouse non redundant nucleotide collection at NCBI. Total RNA was isolated from three different cell preparations for each cell line using RNeasy Midi (Qiagen), followed by DNaseI treatment. cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Roche). All qRT-PCR reactions were performed on a Lightcycler 480 (Roche) using Fast Start SYBR Green Master mix (Roche). The specificity of each amplification reaction and the absence of primer dimer formation were additionally verified via an evaluation of the melting curve of the amplified product and via gel electrophoresis. Amplification efficiencies for each primer set (target and reference genes) were determined on an equivalent mixture of WT, KO, HET and KI MEFs. The absence of amplification from putative contaminating DNA in the RNA preparation was ensured using a control sample of RNA from which no cDNA was synthesized. qRT-PCR reactions on biological triplicates were performed in duplicate on a LightCycler 480 (Roche) using the Fast start SYBR Green master mix (Roche).

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We normalized these values using a normalisation factor calculated by qBASE software [23] (http://medgen.ugent.be/qbase/) that was based on qRT-PCR results of a set of reference genes analysed for each sample. The combination of pgk1 (phosphoglycerate kinase 1), ubc (ubiquitinC), s18 and gapdh as reference genes resulted in the lowest variation of reference gene relative quantities across the samples. The normalized Relative Transcript level was calculated. Adhesion assays The adhesion and contractility assays were performed as previously described [24, 25]. Briefly, 104 cells were seeded in triplicate for 60 min into 96-well plates coated with collagen (10 µg/cm2). After removal of unattached cells, the adherent cells were fixed, stained with crystal violet and quantified by absorbance at 595 nm. Alternatively cells were allowed to adhere for three hours, fixed and processed for immune fluorescence. Transforming growth factor β (TGFβ) activity was measured using the mink lung epithelial cell (MLEC) luciferase assay [26]. Briefly, 2,000 to 3,000 WT or KO1 MEFs were cultured on plastic. On day 4, 20,000 MLECs were added and luciferase activity was measured on day 5. Three independent experiments were done (two tetraplicates and one duplicate). The average fold of TGFβ activation per technical replicate was calculated and these were used to calculate the overall average fold activation of KO1 MEFs and standard error of the mean (SEM). Real-time monitoring of cell adhesion using impedance technology was performed on an xCELLigence plate E device (Roche). The xCELLigence apparatus was run according to the manufacturer instructions. Wells were either not pretreated, coated with 10 µg/cm2 collagen in PBS or coated with 6.25 µg/cm2 fibronectin in DMEM. Each well was seeded with 104 cells and this was done in triplicate for each condition. The impedance was measured continuously for 12 hours and expressed as Cell Index (CI). Immunofluorescence and phase contrast microscopy

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Immunofluorescence and phase contrast microscopy were done with an Olympus inverted XI71 or XI81 microscope. Images were recorded using a CCD digital camera (SPOT-RT monochrome; Progress Control) controlled by the AnalySIS docu software (Olympus) or on an Olympus-CellM system. Fluorescence images were recorded with a 60X LCPlanFL objective (N.A.=0.70, Olympus). Cells were seeded on glass cover slips coated with 10 µg/cm2 collagen and fixed after 3 h or 24 h. For visualizing actin isoforms and vinculin with specific antibodies and

for

monitoring

F-actin

by

phalloidin

staining,

established

procedures

using

paraformaldehyde fixation and Triton-X100 permeabilisation were followed. The primary antibodies used are described above. The secondary antibodies and the phalloidin antibodies are Alexa fluor conjugates (Molecular probes). For ROCK or blebbistatin (Calbiochem) treated samples the cells were first allowed to adhere 5 h and then incubated with 10 µM of inhibitor and fixed after 21 h. Phase contrast images were recorded with a 10X UPlanFL objective (N.A.=0.30, Olympus). Quantitative videomicroscopy for the determination of random 2D cell migration on collagen for 4 h was done as described [27] (for a detailed description of hull area in µm² see Supplementary Material and Methods and Fig. 4 in reference [28]). For ROCK or blebbistatin treatment we used 10 µM of inhibitor. Kymographs were recorded with a 60x OiPH-UPLFLN objective (N.A. 1.25, Olympus). Images were taken every 10 s for 10 min and analyzed with the Kymograph Plugin for ImageJ (http://rsb.info.nih.gov/ij and http://www.embl.de/eamnet). For scratch wound assays we plated 5.104 cells per well of a 48-well dish 24 h before making uniform wounds with the Cell Scratcher (Peira Scientific Instruments). Wound closure was imaged every 15 min for 24 h. The images were analyzed using an algorithm described in [29]. SILAC and differential COFRADIC and mass spectrometry

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COFRADIC proteomics was done as described previously [30, 31]. β-actin WT and β-actin KO cells were grown in culture medium supplemented with

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C514N- or

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C515N-methionine

(Cambridge Isotope laboratories) for at least six doubling times [32]. Cells were collected and flash frozen in liquid nitrogen. Cell pellets containing 2-4 106 cells were lysed in 0.7% CHAPS, 50 mM HEPES, pH 7.4, 100 mM NaCl, complete protease inhibitor cocktail tablet (ROCHE). Lysates were cleared by centrifugation. 250 µg of total protein was processed further. Following trypsin digestion, methionyl peptides were isolated by combined fractional diagonal chromatography (COFRADIC) as described previously [33]. Such isolated peptides were sampled by LC-MS/MS using an Ultimate 3000 HPLC system (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany). Peptides were first trapped on a trapping column (PepMap™ C18 column, 0.3 mm internal diameter (I.D.) x 5 mm length (Dionex)) and following back-flushing from the trapping column, the sample was loaded on a 75 μm I.D. x 150 mm length reverse-phase column (PepMap™ C18, Dionex). Peptides were eluted with a linear gradient of 1.8% solvent B’ (0.05% formic acid in water/acetonitrile (2/8, v/v)) increase per minute at a constant flow rate of 300 nl/min. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant ion peaks per MS spectrum. Full scan MS spectra were acquired at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap. In the LTQ, MS/MS scans were recorded in centroid mode at a target value of 5,000 ion counts. Peptides were fragmented after filling the ion trap with a maximum ion time of 10 ms and a maximum of 1E4 ion counts. From the MS/MS data in each LC run, Mascot generic files (mgf) were created using the Mascot

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Distiller software (version 2.2.1.0, Matrix Science) and were searched against the SwissProt database (v14.07) restricted to Mus musculus taxonomy (15813 entries) using the Mascot search engine (version 2.2.1). The following MASCOT parameters were set: the protease setting was trypsin (cleavage between Lys or Arg and Pro was tolerated) with a maximum of one allowed missed cleavage, whereas acetylation of a protein’s N-terminus, deamidation of Asn and Gln, formation of carbamidomethyl Cys, oxidation of carbamidomethyl Cys and pyroglutamate were considered as possible modifications, and tolerances for the precursor ion mass and fragment ions masses were set to ± 10 ppm and 0.5 Da respectively. Determination of the light (12C514NMet) and heavy (13C515N-Met) labeled sulfoxide methionyl peptides for further quantification was established by using the quantitation option in Mascot. Finally, peptide hits of which the MASCOT ion score of the MS/MS spectrum exceeded MASCOT’s identity threshold score set at 99% confidence and which were ranked one were withheld and considered identified. An FDR of 0.25% was determined by the method described by Käll et al. ([34]). The total list of identified peptides is available via the PRIDE data repository (http://www.ebi.ac.uk/pride/; PRIDE experiment accession number: 18462-18463 ([35]) and includes the % coverage and the number of different peptides per protein (see also Suppl_Table_PRIDE_18462 and Suppl_Table_PRIDE_18463). Quantification was performed using Mascot distiller software (version 2.2.1.0, Matrix Science). Ratios are calculated from a least square fit of the area below the light and heavy isotopic envelope situated in the elution peak of the precursor determined by the Distiller software (XIC threshold 0.3, XIC smooth 1, Max XIC width 250). To validate the calculated ratio, the standard error on the least square fit has to be below 0.16 and correlation coefficient of the isotopic envelope should be above 0.97. All data management was done by ms_lims ([36]). Only these proteins which were quantified by at least 2 different peptides, were

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used for quantification (Supplemental Table 2C). If more than one MS/MS spectrum was linked to a peptide, the mean of all its calculated ratios was considered as the ratio value for that peptide. The average ratio values of different peptides representative for a protein were used as the ratio value for altered expression of that protein. Robust statistics [37] was applied to the base-2 logarithm of the ratios to identify proteins that significantly deviate from the median ratio. For both conditions WT/KO1 and WT/KO2, a skewed normal distribution with Huber scale of 0.68 and 0.88 on a 95% confidence level respectively and a median of respectively non log 1.112 and 1.105 (Supplemental Table 2C). For pathway analysis (see below) we only considered the intersection of proteins that were deregulated in both KO cell lines. Since these are derived from littermates the intersection can be considered as being derived from two biological replicates. Expression levels of selected proteins were validated by standard qRT-PCR or WB or both (see above and Supplemental table 5 for a guide to the respective figures). Robust statistics to identify the over represented pathways and Ingenuity Pathway Analysis To identify regulated pathways, robust statistics [37] was applied. The validated quantitative proteomics data were subdivided in three. The first subdivision contained the proteins that were upregulated in the heavy configuration (L/H < 0.5, in β-actin KO MEFs). The second subdivision holds the proteins that were downregulated in the heavy configuration (L/H > 2). The last subdivision contains the rest of the proteins and were considered as unchanged protein levels (0.5 < L/H < 2). The p-value for every pathway found in the different parts was calculated via the hyper geometric test, using the proteome size (the number of proteins in the proteome that are linked to a KEGG pathway), the sample size (the number of proteins in the subdivision that are linked to a pathway), the in-proteome pathway links (the number of proteins that are linked to a specific pathway) and the in-sample pathway links (the number of proteins in the

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subdivision that are linked to the same specific pathway). A pathway was considered regulated if the calculated p-value for one subdivision was larger than 0.975 or smaller than 0.025. Only KEGG pathways that were regulated in both KO cell lines derived from the first subdivision (condition L/H