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Application of High-Content Analysis to the Study of Post-Translational Modifications of the Cytoskeleton Peter J. M. Drake,†,# Gareth J. Griffiths,‡,# Leila Shaw,† Rod P. Benson,‡ and Bernard M. Corfe*,#,† Human Nutrition Unit, School of Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom, and Imagen Biotechnology Ltd., Grafton Street, Manchester M13 9NT, United Kingdom Received August 14, 2008

Cytokeratins 8 and 18 have recently been identified as acetylated. The acetylation of other cytoskeletal proteins has been reported as linked to stabilility. As colorectal cells exist bathed in pharmacologically active levels of the HDACi butyrate, we sought to apply state-of-the-art High Content Analytical approaches to identify the effect of butyrate upon the cytoskeleton of colorectal cells. We observed butyrate caused an increase in acetylation at three distinct residues of cytokeratin 8 (K10, K471, and K482) and that the kinetics of altered acetylation were distinct, implying either separate HDACs, or a heirachy of acetylation. This change in acetylation was associated with a breakdown in the cytokeratin cytoskeleton, implying a functional role for cytokeratin acetylation. Keywords: High Content Analysis • Keratin • Acetylation • HDAC • Butyrate

Introduction High-fiber diets have been shown to have an inverse relationship with the occurrence of colorectal cancer.1 One potential mechanism for fiber’s chemopreventive action is through the influence of short-chain fatty acids (SCFAs) on epithelial tissue. SCFAs are produced by the anaerobic fermentation of fiber by symbiotic bacteria in the colon. While the SCFA butyrate is a preferred nutritional source for colonocytes,2 it is its effect on key cellular processes which appears to be the major influence on reducing neoplasmic occurrence. Butyrate has been shown both in vitro and in vivo to alter cell cycle, cell differentiation and cell death.3 These alterations in cell fate are consequent to its function as an inhibitor of histone deacetylases (HDACs) at physiological concentrations. HDACs play a role in maintaining a balance of acetylation of histones in opposition to a group of enzymes called histone acetyltransferases (HATs). Alterations in histone acetylation result in widespread changes in transcription across the genome.4,5 It is also increasingly being shown that HDACs have substrate proteins other than histones whose function is alterable by deacetylation. Other proteins regulated through acetylation include nuclear structural proteins,6 transcription factors such as Sp17 and the guardian of the genome, p53.8 One specific type of protein whose components are subject to HDAC deacetylation is cytoskeletal protein. R-Tubulin is subject to the actions of HDAC6 and SirT2, two cytoplasmic deacetylases. 9,10 Of the HDACs, HDAC7 is also found in the cytoplasm.11 HDACs 4 and 5 are reciprocally cytosolic during * To whom correspondence should be addressed. E-mail: b.m.corfe@ sheffield.ac.uk. † University of Sheffield. ‡ Imagen Biotechnology Ltd. # These authors contributed equally to this work.

28 Journal of Proteome Research 2009, 8, 28–34 Published on Web 11/05/2008

myocyte differentiation.12 In the epithelium, the cytoskeletal component protein of the intermediate filaments are composed of polymerized dimers of type 1 and 2 keratin. In intestinal epithelium, CK8 (type 1) and CK18 (type 2) are the primarily expressed proteins and gene ablation of CK8 in mice has been shown to cause high incidence of inflammation and hyperplasia in mice reaching adulthood.13 Human heritable predispositions to ulcerative colitis have been mapped to the CK8/ 18 loci 14 and CK8 mutations have been implicated in early epithelial carcinogenesis: two isoforms of CK8 were among a group of proteins recognized as being up-regulated in pericancerous areas in colorectal adenoma and down-regulated in the adenoma itself.15 Keratin 8 has been implicated in apoptosis regulation, cellular regularity in cell death regulation and tumor necrosis factor-R and Fas-related cell-death signaling pathways in apoptosis. The absence of CK8 and 18 has been shown to increase epithelial cell sensitivity to TNF in apoptosis. Our work has shown that, like microtubules, the keratins in intermediate filaments are acetylated.16 We reported mass spectrometric identification of five acetylation sites on keratin 8 and one on keratin 18. Our data suggested that HDACs 5 and 8 copurified with IF and were therefore potential keratin deacetylases. Western blotting also showed butyrate increases the apparent fraction of CK8 in the acetylated form, thus, implying that not only is CK8 a likely substrate for HDACs, but that butyrate is an inhibitor of the enzyme(s) concerned. Immunocytochemical analysis of Caco-2 cells has also shown that butyrate has an effect on the structure of the intermediate filaments formed within the cell, visibly reducing their filamentous nature with increasing concentration (MacKellar and Corfe, unpublished results). High-content analysis (HCA) is a tool extensively used within the pharmaceutical industry and is based on the computational analysis of images captured by an automated fluorescence 10.1021/pr8006396 CCC: $40.75

 2009 American Chemical Society

Cytokeratin Acetylations Respond to Butyrate

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Figure 1. Treatment of Caco2 cells with butyrate slows mitosis. (A) Image of cells treated with butyrate showing mitotic figures. Arrows indicate dysmorphic mitotic nuclei. (B) Caco 2 cells were treated for 20 h with various doses of butyrate (abscissa) and then the cells fixed in ice-cold 70%methanol. The cell nuclei were used to define the cell regions of interest and calculate the total cell number from the 15 fields of view. Each data point represents the mean ( SEM of three separate experiments. (C) The cells were rescanned on the arrayscan using the compartmental analysis algorithm set to detect an increase in nuclear intensity that is compatible with mitosis (see Experimental Details). Data points represent the mean ( SEM of three plate repeats.

microscope adapted to read microtiter plates. There are several advantages to HCS over traditional fluorescence microscopy, Western analysis and ELISA assays. First, HCS allows the quantification of staining intensities and subcellular localization patterns allowing image data to be converted into a graphical format. Second, unlike ELISA or Western analysis, the information is collected on a cell by cell basis. Third, the use of microtiter plates means that many more treatment conditions can be analyzed than is possible with either traditional fluorescence microscopy or Western analysis. Finally, the rapid acquisition of multiparametric data from microtiter plates means that the cost savings are substantial because both the number of person hours involved in performing the experiment and the amount of laboratory consumables used are greatly reduced. HCA has many uses particularly in drug development and has streamlined the amount of work that is required to validate drugs before animal and clinical testing become necessary. An area where HCA has had an important impact is in the use of small inhibitory RNA (siRNA) to identify genes as potential drug targets on the basis of a desired alteration in cellular function in response to the gene’s inactivatation by downregulation of its transcription.18 This type of analysis is particularly helpful in cancer research where genes are suspected to be carcinogenic when mutated or inactivated. Toxicology studies are able to utilize analyses of, for example, membrane permeability and nuclear size (two indicators of toxicity of drug treatments) to eliminate drug candidates in fixed and kinetic (live) assays; giving HCA an edge over some cytometry technology which cannot yield kinetic or long-

duration analyses. Apoptosis, cell cycle and cell-viability studies are possible with cell cycle phase identification software. It is also possible to study proteins such as ion channels, their receptors and their internalization by way of the distribution of proteins on membranes, in cytosol and in the nucleus. Several recent reviews indicate the scope and potential of HCA for drug discovery programs19-21 Finally, the rich independent and dependent variable space that HCA offers makes it a natural experimental companion to the discipline of System Biology and it is for these reasons that the use of HCA is rapidly growing within the pharmaceutical industry and to a lesser extent academia.22,23 Like phosphorylation, acetylation can be studied as a quantifiable post-translational protein alteration, with many function-specific consequences, for example, in cytoskeletal integrity and cell viability. With the use of HCA-specific techniques and protocols almost identical to those of ICC, it is possible to locate and quantify the intensity of immunofluorescent antibodies and to measure the location and dispersion of cellular protein expression within the cell and in relation to the rest of the cellular structure. The technology can also be applied at a histological level to gauge the effects of a treatment in colonies of cells, such as distribution of a protein in the periphery of a colony compared to more central, confluent (differentiating) cells or the size of colonies in clonal studies; and with some machines the ability to take confocal images to better understand and more clearly compose the 3D structure of the protein within the cells. We have combined newly generated, acetylation-specific antibodies to keratin 8 epitopes Journal of Proteome Research • Vol. 8, No. 1, 2009 29

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Figure 2. Antiacetyl antibodies to CK8 acetylations colocalize with CK8. Caco2 cells were fixed with methanol and immunostained with anti-CK8 antibodies (green) and anti-acetylation antibodies (red, sites as indicated). Nuclei were localized with Hoechst. Cytosolic immunostaining with the three acetyl-specific antibodies colocalized with CK8 as indicated by the yellow staining in the merged image; however, there were some nonspecific cross-reactions in the nucleus, where no CK8 was observed.

with an HCA-driven approach to address key questions on the role of different acetylations within the cell during cell cycle and proliferation and in response to the physiologically relevant HDACi, butyrate.

Experimental Details 1. General Reagents and Solutions. Phosphate-buffered saline Dulbecco “A” solution (PBS) was supplied by Oxoid, Basingstoke, U.K. DMEM (Dulbecco/Vogt-modified Minimal Essential Medium) with 1 g/L D-glutamine, 4 mM L-glutamine, 110 mg/L sodium pyruvate and 25 mM HEPES was supplied by Gibco Life Technologies (Invitrogen, U.K.). Added to the DMEM was 5% (v/v) penicillin (10 000 units · mL-1) and streptomycin (10 000 µg · mL-1, both from Gibco Life Technologies, U.K.), and 10% (v/v) heat-deactivated fetal calf serum (FCS Biosera, Sussex, U.K.). Ethanol and methanol were supplied by Fisher Chemicals (U.K.). HCT-116 colon carcinoma cell line and Caco2 epithelial carcinoma cell line are laborarory stocks. All cell lines used in this laboratory are screened bimonthly for mycoplasma contamination. No mycoplasma was detected in these cells or any other in the laboratory during this study 2. Cell Culture Protocol. HCT116 cells and Caco-2 cells were cultured in DMEM, and incubated at 37 °C, 5% CO2 in humidified air, and treated with either sodium butyrate (NaB, Calbiochem) or nicotinamide (Sigma) 3. Protocol for HCA Analysis. 3.1. 96-Well Plate Seeding. HCT-116 and Caco-2 cell-lines were suspended in 30

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DMEM at 6.25 × 104 cells · mL-1 and 5.0 × 104 cells · mL-1, respectively. The suspended media was transferred 200 µL per well into black-sided Costar 96-well plates treated for cell culture (Sigma-Aldrich, U.K.). Cells were incubated for 48 h. 3.2. Cell Treatment. Cells were treated with either sodium butyrate (NaB, Calbiochem) or nicotinamide (Sigma) at concentrations indicated for 24 h. Cells were fixed with -20 °C 100% methanol for 5 min and washed twice in PBS to be left to sit in 200 µL of PBS and sealed (sealing film from Greiner). 3.3. Immunocytochemistry. All antibodies were diluted in digitonin according to Cellomics’ HCA protocols. Antibodies used were Tubulin, Keratin 8 (Abcam) K10 (raised in rabbit to acetylation at lysine 10 of keratin8), K471 (raised in chicken to acetylation of lysine 471 of keratin 8) and K482 (raised in rabbit to acetylation of the C-terminal lysine of keratin 8). The K10, K471 and K482 antibodies were raised specifically for this study. Antibody validations are shown in the Supporting Information. Secondary antibodies used were anti-mouse (Alexa fluorophore 488, green), anti-rabbit and anti-chicken (both Alexa fluorophore 555, red). DNA was stained with Hoechst 33342 at 2.5 µg · mL-1. Fixed, immunostained cells were then stained with Cellomics Whole-Cell Red Stain to visualize cell shape and cytosolic area. Cells were stored in PBS and sealed prior to analysis. 3.4. High-Content Analysis/Screening. HCA was undertaken using a Cellomics ArrayScan II, version 3.5.1.2. Cellomics’ proprietary software was used; the algorithms used for the

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Cytokeratin Acetylations Respond to Butyrate

Figure 3. Quantification of cytosolic staining intensity for analysis. The left panels show staining in untreated cells where nuclei are stained blue and acetylation antibodies are stained red. The center panels show images from the top dose of butyrate used in these experiments (10 mM). An increase in overall intensity is evident in both the cytoplasm and the nucleus. The panels on the right are overlaid with computer generated pixels (purple) which correspond to the signal detected by the algorithm. From the purple overlay, it is evident that only cytosolic staining is being quantified using the algorithm setup.

analysis were Morphology Explorer and Compartmental Analysis. With the use of the Compartmental Analysis algorithm, a mask was created so that only cytoplasmic staining was measured thereby excluding any nuclear fluorescent signal. The data generated measured the total staining intensity of CK8, acetylated K10, acetylated K471 and acetylated K482. To obtain a texture measurement for the staining, the morphology algorithm (Cellomics) was employed to calculate a co-occurrence intensity measurement. This is a texture measurement derived from the probability of pixels with different intensities occurring next to each other. Where the staining is uniform, the texture intensity measurement is low. Conversely, highly varying intensities, indicative of structural staining, result in a larger value for this parameter. The Compartmental analysis algorithm was also used for calculating numbers of mitotic cells in the population. With the use of the Average Nuclear Intensity parameter, it is possible to create a gate so that nuclei with intensely packed

mitotic DNA can be quantified. Confounding apoptotic cells are excluded from this analysis by setting a minimum DNA content threshold.

Results 1. Butyrate Causes Increased Numbers of Mitotic Cells in Culture. We and others have previously reported that butyrate causes altered passage through the G2/M phase of cell cycle. 24,25 A particular advantage of the Cellomics HCA over competing methodologies is the facility to measure large numbers of mitoses in a cell population objectively. This is not possible by flow cytometry owing to the lack of difference in DNA content between G2 and M, and is time-consuming and subject to observer bias when scored by microscopy. We therefore sought initially to use this system to analyze whether butyrate can specifically alter passage through M phase of cell cycle. Caco2 and HCT116 colon epithelial cells were treated with a range of butyrate concentraJournal of Proteome Research • Vol. 8, No. 1, 2009 31

research articles tions from 0 to 20 mM for a period of 20 h. Microscopic analysis showed elevated numbers of mitotic figures in both cell lines with treatment with butyrate, most noticeably at the higher concentrations (Figure 1C). Automated cell counts revealed small decreases at 20 h of treatment with higher concentrations but did not have substantial effects on cell number (Figure 1B), in line with previous findings which showed a reduction in cell count attributable to cell death at around 48 h after treatment.26,27 We have previously shown that there is no loss in cloning efficiency of cells with 20 h butyrate treatment,26 so we infer that this mitotic blockade may be removed following butyrate withdrawal. The data shown are subject to three independent repeats, each with internal triplicates. Taken together, the data show alteration in cell cycle triggered by butyrate is associated with impaired mitosis. Review of acquired images (Figures 1A) reveals dysmorphic mitotic nuclei which we speculate may be associated with impaired microtubule organization. 2. Novel Antiacetyl-CK8 Antibodies Reveal Acetylation Throughout the Intermediate Filament Cytoskeleton. We have recently reported the identification of five novel acetylation sites on cytokeratin 8. Two of these sites occur within the coiled-coil domains of the protein, and three at the globular N- and C-termini of the protein,16 regions of the protein known to have post-translational modifications involved in the regulation of intermediate filament assembly. Antibodies to the three acetylations outwith the coiled-coil domains were raised in rabbit (K10 and K482) and chicken (K471). Antibodies produced a single cross-reactive band at the correct molecular weight for CK8 on Western blot of intermediate filament preparation (data not shown). Antibody concentrations for immunocytochemistry were established and dual-staining studies undertaken. Figure 2 reveals the staining patterns for all three acetylCK8 antibodies as against CK8 itself with images captured by the Cellomics Arrayscan. All three antibodies show a staining pattern in the cytosol which is broadly filamentous. Dual staining with the CK8 antibody reveals that the acetyl-CK8 cytosolic staining colocalizes with CK8. Taken together with the validation studies from Western blotting, we are confident that this cross-reaction is specific to CK8 acetylations. We did note, however, that two of the antibodies produced a significant nonspecific cross-reaction in the nucleus. We assume that this is due to the abundance of acetylations in the nucleus, for example, histones and nuclear structural proteins. 3. Quantification of Cytosolic Cross-Reaction. With the use of the Arrayscan compartmental analysis algorithm, it was possible to create two subcellular regions of interest representing the cytosol and the nucleus, respectively. Thus nonspecific staining in the nucleus can be excluded from the cytoplasmic analysis. Figure 3 shows cells stained for each of the three different acetylation sites. The left panels show staining in untreated cells where nuclei are stained blue and acetylation antibodies are stained red. The center panels show images from the top dose of butyrate used in these experiments (10 mM). An increase in overall intensity is evident in both the cytoplasm and the nucleus. The panels on the right are overlaid with computer generated pixels (purple) which correspond to the signal detected by the algorithm. From the purple overlay, it is evident that only cytosolic staining is being quantified using the algorithm setup. 4. HCA Reveals Alteration in Acetylations in Response to Butyrate Which Are Associated with Breakdown of the IF Cytoskeleton. Butyrate is a physiologically relevant HDACi known to have a key role in regulating cell division and 32

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Figure 4. The acetylation of cytokeratin 8 in response to increasing doses of Butyrate. Caco 2 cells were treated for 20 h with varying doses of butyrate (abscissa) before fixing in ice-cold methanol. The cells were then treated with an antibody raised to a particular cytokeratin 8 acetylation site: K10 (black, circles), K471 (green, squares), K482 (red, triangles) and the binding of this antibody was detected with a secondary antibody conjugated to Alexa-555. Cell nuclei were stained with Hoechst 33342 and High Content analysis was performed on a Cellomics Arrayscan using the nuclear dye to identify cell regions of interest. The intensity of Alexa-488 staining was then quantified and all the data normalized to the 0 dose control. Each data point represents the mean ( SEM of three individual experiments.

apoptosis in the colon. As cytokeratins are thought to be involved in the processes of apoptosis and anoikis, we hypothesized that butyrate may alter the integrity of the cytoskeleton during apoptosis through alteration in protein acetylation. Caco2 and HCT116 cells were treated with increasing concentrations of butyrate from 0 to 10 mM for 20 h. The amount of cytosolic fluorescence associated with K10, K471 or K482 crossreaction was analyzed in each cell line at each concentration. We noted that each acetylation increased in response to butyrate (Figure 4). These alterations are significant. Critically the concentration-response curve for each is distinct. K10 acetylation increases markedly between 2 and 5 mM butyrate treatment, being reasonably stable to lower concentrations. This increase is ca. 2.5-fold over the levels of acetylation quantifiable in untreated cells. Similarly, K471 acetylation increases in response to elevated concentrations of butyrate, but notably is only increased at 10 mM. The increase in fluorescence is similarly ∼2.5-fold over basal levels. Acetylation of K482 again increases in response to butyrate but again has a distinct profile. There is a clear concentration-response curve across the whole range of butyrate treatment with K482 acetylation. 5. The Effect of Butyrate on Cytoskeletal Integrity. The degree of CK8 filament-like staining was determined in Caco2 and HCT116 cells treated with various concentrations of butyrate. Cells were treated with butyrate as described above, immunocytochemically stained for CK8 and examined microscopically (Figure 5A). Untreated cells showed a characteristic filamentous architecture within the cells; however, after treatment, the staining pattern became diffuse and pan-cytosolic. The degree of filament-like staining was quantified using the arrayscan morphology algorithm (Cellomics). This program can produce an intensity concurrence measure which is proportional to the amount of fine structure (texture) within the cell.

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Cytokeratin Acetylations Respond to Butyrate

Figure 5. The measurement of cytokeratin 8 filamentousness. (A) Caco2 cells treated or otherwise with 5 mM butyratre for 20 h and immunostained for cytokeratin 8. After treatment with butyrate, the staining pattern si diffuse and pan-cytosolic in contrast to the filamentous organized structure of staining prior to butyrate treatment. (B) Cytokeratin 8 immunostaining in cells treated for 20 h with various doses of butyrate (abscissa) before fixing in 1% formaldehyde was detected using a secondary antibody conjugated to Alexa-488. The analysis algorithm examines the variance in pixel intensity surrounding a given pixel within a defined spatial grid. The higher the variance, the higher the “texture intensity” score. A highly variant pixel range within a small area represents what visually would be described as a “rough looking” staining profile, while highly correlated pixel values within a given area results in a staining profile that visually looks smooth. Each data point represents the mean ( SEM of three experiments.

ingly recognized as an important post-translational modification of proteins, paralleling phosphorylation in its potential to regulate protein function. We have generated acetyl-specific antibodies to three sites on CK8 and we report here the application of these antibodies to study of IF function. All three antibodies cross-react with a protein of the same molecular weight as CK8 in IF preps and colocalize with CK8 fluorescence in immunocytochemical analysis (not shown and Figure 2). Taken together, these data suggest that the cytosolic cross-reactivity is specific to CK8 acetylation. Treatment with butyrate caused elevation of acetylation at all three acetylation sites and this occurred in a concentration-responsive manner. The concentration-response curve for each increase in acetylation was distinct: K482 showed a distinct increase at low concentrations of butyrate (0.5 mM) which increased up to 10 mM. In contrast, both K10 and K471 were only elevated at higher concentrations, with K10 elevated above 5 mM and K471 above 10 mM (Figure 4). Three distinct models may account for this observation: (i) the three acetylation sites may be regulated by different HDACs with different inhibition profiles in response to butyrate; (ii) the acetylation of CK8 may be a stepwise process where K482 acetylation is required prior to K10 acetyltation which in turn precedes K471 acetylation; (iii) the acetylation sites may themselves be unaltered but subject to a conformational alteration in CK8 associated with breakdown of filaments (Figure 5) which exposes the epitope. Accelerated Discovery through Application of HCA. The data described in the current work clearly demonstrate the power of automated fluorescence microscopy to address complex biological questions. With the use of High Content Analysis, we were able to examine the acetylation of CK8 at 3 distinct sites and clearly map the individual response of each site to increasing concentrations of butyrate. Furthermore, we were also able to elucidate subtle morphological changes that were occurring to the cytoskeleton in response to butyrate and which may ultimately be linked to the functioning of CK8. Finally, by rescanning the plates with a different analysis algorithm, we were able to obtain the percentage of cells in mitosis and so examine a critical cellular response parameter simultaneously with the CK8 parameters under study.

Conclusion High Content Analysis (HCA) has provided an accelerated, sensitive and highly quantifiable discovery rate for analysis of post-translational modifications of the cytoskeleton.

Acknowledgment. This work was supported by the University of Sheffield Office of Corporate Partnerships. Supporting Information Available: Figure of antibody validations. This material is available free of charge via the Internet at http://pubs.acs.org. References

We found a significant decrease in the concurrence measure, especially evident at concentrations of butyrate above 2 mM, indicative of smoother less filament-like staining of CK8.

Discussion Spatial Proteomics of CK8 Acetylation. We have previously reported the acetylation of CK8 at multiple sites. The role of these acetylations was unclear; however, acetylation is increas-

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