Synonymous Modification Results in High Fidelity Gene Expression of Repetitive Protein and Nucleotide Sequences

Transcription

1 Synonymous Modification Results in High Fidelity Gene Expression of Repetitive Protein and Nucleotide Sequences Bin Wu *,1,2, Veronika Miskolci *,1, Hanae Sato 1, Evelina Tutucci 1, Charles A. Kenworthy 1, Sara K. Donnelly 1,2, Young J. Yoon 1, Dianne Cox 1,2, Robert H. Singer 1,2,%, and Louis Hodgson 1,2,% 1 Department of Anatomy and Structural Biology, 2 Gruss-Lipper Biophotonic Center, Albert Einstein College of Medicine, Bronx, NY Contents Supplementary Figures Figure S1, Two-color FISH image of transiently transfected 24xMS2SL mrna Figure S2, Histogram of fluorescence intensity of FISH spots of 24xMS2SL. Figure S3, Protease inhibitor treatment does not rescue the apparent biosensor degradation effects of Rac1bs(ori) in RAW264.7 cells. Figure S4, Sequence alignment between the original versus the synonymously modified mcerulean1 and the binding domains used in the single-chain biosensor for Rac1 GTPase. Figure S5, Biosensor truncation after selection for stable integration following plasmid transfection. Supplementary Movies

2 Fig. S1. Two-color FISH image of transiently transfected 24xMBS-SL mrna. CFP-24xMBS-SL was transiently transfected in U2OS cells. Two color FISH image was performed in a, red: CFP, green: MBS-SL, scale bar: 5µm. The box was enlarged in b, the scale bar: 2µm, top, merged, middle: CFP, bottom: MBS-SL.

3 Fig. S2. Histogram of fluorescence intensity of FISH spots of 24xMBS-SL. The stably expressed mrnas CFP-24xMBS-SL were detected in the ORF channel by FISH Quant. The corresponding fluorescence intensities in the MBS channel were measured. The histogram of fluorescence intensities of MBS channel were plotted for cells with colocalization efficiency larger than 0.8 (a) or less than 0.2 (b). The FISH intensity in (b) was lower than that of the (a).

4 Fig. S3. Protease inhibitor treatment does not rescue the apparent biosensor degradation effects of Rac1bs(ori) in RAW264.7 cells. PI: Protease inhibitor cocktail (Sigma P1860 DMSO solution) was used at 1:250 dilution in culture during the 24 hr induction of the original version of the Rac1 biosensor in RAW264.7 cells.

5 Fig. S4. Sequence alignments between the original versus the modified monomeric Cerulean1 and the binding domains used in the single-chain biosensor for Rac1 GTPase. Monomeric Cerulean1 and the p21 binding domain (PBD) of the p21 activated kinase 1 were codon

6 optimized to achieve approximately 66% sequence homology with the monomeric Venus and the second PBD, respectively, in the single-chain biosensor for Rac1 GTPase.

7 Fig. S5. Biosensor truncation after selection for stable integration following plasmid transfection. (a) The biosensor for Wiskott Aldrich Syndrome protein (WASp) published previously (Cammer et al. 2009). This biosensor is based on FRET between ECFP and EYFP, attached onto the N/Ctermini of the full-length WASp. (b) Biosensor expression in stable cell line (RAW264.7) shows truncated product at approximately 100kDa. Lane 1: Parental untransfected; Lane 2: shwasp-stable, biosensoruntransfected; Lane 3: shwasp-stable, biosensor-stable from transfection and selection. BS : fulllength biosensor; Endo.: endogenous WASp; Trunc.: truncated product. Because of this truncation problem encountered, this biosensor was used only in transient transfections in the previous publication (Cammer et al. 2009).

8 Supplementary Movies Supplementary Movie 1, Live cell imaging of 24xMBSV5: U2OS cell stably expressing mcherry- 24xMBSV5 and stdmcp-stdgfp were streamed with 50ms exposure time for 50 frames. Supplementary Movie 2, Representative live cell imaging of the Rac1(ori) biosensor stably and inducibly transduced in RAW264.7 macrophage cell line, in serum-stimulated random protrusions. Cells were imaged under 60x magnification 2x2 binning, FRET and mcer fluorescence emission channels were acquired simultaneously on two cameras to eliminate motion artifacts. Images were taken every 10 sec. Supplementary Movie 3, Representative live cell imaging of the Rac1(syn) biosensor stably and inducibly transduced in RAW264.7 macrophage cell line, in serum-stimulated random protrusions. Cells were imaged under 60x magnification 2x2 binning, FRET and mcer fluorescence emission channels were acquired simultaneously on two cameras to eliminate motion artifacts. Images were taken every 10 sec. Cammer M, Gevrey JC, Lorenz M, Dovas A, Condeelis J, Cox D The mechanism of CSF-1-induced Wiskott-Aldrich syndrome protein activation in vivo: a role for phosphatidylinositol 3-kinase and Cdc42. J Biol Chem 284: