Reduced Expression of Lamin A/C Results in Modified Cell Signaling and Metabolism Coupled with Changes in Expression of Structural Proteins Songbi Chen, Catherine Martin, Apolinar Maya-Mendoza, Chi W. Tang, Josip Lovric´, Paul F. G. Sims, and Dean A. Jackson* Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom Received June 23, 2009
Nuclear lamins are intermediate filament proteins that define the shape and stability of nuclei in mammalian cells. In addition to this dominant structural role, recent studies have suggested that the lamin proteins also regulate fundamental aspects of nuclear function. In order to understand different roles played by lamin proteins, we used RNA interference to generate a series of HeLa cell lines to study loss-of-function phenotypes associated with depletion of lamin protein expression. In this study, we used genome-wide proteomic approaches to monitor global changes in protein expression in cells with 2000 proteins that make up the bulk of the total HeLa cell and nuclear proteomes fewer than 2% show significant differences in expression. It must be noted however that many proteins, and in particular modified post-translational isoforms, will not be present in sufficient quantities to allow visualization and identification by routine staining of protein spots in 2D gels. Because sensitivity will clearly limit the analysis of complex protein mixtures, we also explored 5202
Journal of Proteome Research • Vol. 8, No. 11, 2009
the use of antibody microarrays to evaluate changes in protein expression in lamin A/C depleted cells. On the basis of the changes in expression of cytoskeletal proteins detailed above and growing evidence that integrated cellular networks regulate cell signaling and proliferation, we used an antibody microarray that specifically targets proteins involved in the control of cell proliferation (Figure 6). This approach offers at least 2 specific advantages: first, it allows identification of proteins involved in proliferation control that might be expressed at levels below that detectable by 2-DE analysis; second, it facilitates expression profiling of different protein isoforms and allows analysis of functionality that is linked to post-translational protein modifications involved in cell cycle control.35,39,40 Soluble protein extracts were prepared from paired normal and lamin depleted cells using the lamin A/C depleted clone Ld27. Paired samples were labeled separately with either Cy3 or Cy5 and antibody arrays incubated with a mixture containing equal amounts of each labeled extract (10 µg/mL). After incubating to allow binding, soluble protein was removed by washing and the arrays scanned to monitor relative protein expression using the Cy3/Cy5 ratio at each position on the array (Figure 6). Using GenePix Pro 6.0 analysis software, we searched for differential protein expression of >1.5-fold (p < 0.05) after averaging samples from three replicated slides from each of three separate extract preparations (Supplementary Table S2
The Proteome of Lamin A/C-Depleted Cells
research articles
Figure 3. The proteome maps of lamin A/C-depleted HeLa nuclei. Comparison of HeLa nuclear proteomes (A and B) prepared from nuclear extracts of untreated HeLa (Control) and lamin A/C depleted clone Ld27 (Ld27). Equal amounts (180 µg) of protein extract were separated by 2-D PAGE (first dimension, 18 cm IPG strips with linear pH 4-7 (A) or 6-11 (B) gradients; second dimension, 12% SDSPAGE) and visualized by staining with colloidal Coomassie. Delta2D gel analysis software was used to compare spot intensities and highlight individual spots with >1.5-fold increase or decrease in expression in clone Ld27 relative to control. Typical examples are indicated (numbered arrowheads). Gels shown are representative of typical experiments performed on at least 6 occasions from at least 3 biological replicates.
shows fold changes of all proteins). Using Treeview software, a clear distinction between lamin A/C depleted and normal samples enabled the clustering into two main groups of protein expression, with expression either up-regulated (in red) or down-regulated (in green) in lamin depleted cells (Figure 6A). To ensure reliability of the microarray binding, the microarray data was validated by immunoblotting (Figure 6B). The representative images shown confirm the depletion of lamin A/C expression and the equivalence of expression of β-actin and R-tubulin. When these control genes were used as normalization controls, microarray analysis identified 13 proteins (Table
3) that were differentially expressed in lamin A/C-depleted cells. In the panel of proteins represented on this microarray, the most significant changes involved
