Journal: bioRxiv

Article Title: Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions

doi: 10.1101/2022.10.22.513249

Figure Lengend Snippet: (a) Schematic of LAB construct used in HEK293T cells. Fragment of Split-TurboID (N) and Arabidopsis protein CIB1 were fused onto prenylated plasma membrane targeted Enhanced Green Fluorescent Protein (pmEGFP) that localizes to the plasma membrane (CibN). The complementary Split-TurboID (C) fragment was fused to blue-light receptor protein Cryptochrome 2 (CRY2) and mCherry fluorescent protein (CryC) and expressed in the cytoplasm. When exposed to blue light, CRY2 interacts with CIB1 and reconstitutes the split-TurboID, which generates reactive biotin for proximity labeling. Both constructs were coexpressed in HEK293T cells. (b) Confocal images show CibN localizing to the membrane and CryC diffused in the cytoplasm of the cells. When excited with a 488 nm laser, CryC localized to the membrane in seconds and dissociated completely in about 600 sec. Scale bar 3 μm. (c) A region of the membrane was excited with 488 nm laser and CryC accumulation was monitored. CryC accumulated at the membrane region in sub-seconds. Scale bar 3 μm. (d) CryC and CibN intensity was measured across the yellow line in (c) with vertical black dashed lines delineating the membrane on the graph. The fluorescence intensity measurements showed strong correlation between CryC and CibN after light exposure. (e) Integrated CryC (red) and CibN (green) intensity was measured in the yellow box in (c) over time, with the vertical black dashed line indicating 488 nm laser excitation. CryC fluorescence reached the maximum intensity within seconds, while CibN fluorescence remained constant.

Article Snippet: CibN was generated by restriction digesting a CIB-pmEGFP plasmid (Addgene plasmid # 28240, ( )) at AgeI and NheI sites and fusing PCR amplified CIB1 and spTN (Addgene plasmid # 153002, ( )) using Gibson assembly.

Techniques: Construct, Clinical Proteomics, Membrane, Labeling, Fluorescence

Journal: bioRxiv

Article Title: Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions

doi: 10.1101/2022.10.22.513249

Figure Lengend Snippet: Ecad-EGFP fused with Split-TurboID(N) and CIB1 (ECibN) and CryC were co-expressed transiently in MDCK cells. Cells incubated with 100 μM biotin were illuminated with blue light, for an hour, on a 10 min on/off cycle. The cells were fixed and stained for EGFP, mCherry and biotin. (a) Schematic of LAB fused with Ecad. EGFP, fragment of Split-TurboID (N), and CIB1 were fused onto the C terminus of Ecad to create ECibN. CryC was expressed in the cytoplasm. Both constructs were co-expressed in MDCK cells. (b) ECibN localizes to the intercellular junction, and in the presence (+) of light and biotin, CryC translocates to the membrane and dimerizes, which results in the biotinylation of proteins closer to the membrane, which is confirmed with composite images showing the colocalization of CibN, CryC, and Sta. In the absence (-) of light (c), biotin (d), or both (e), biotinylation is not observed. When only CryC (f) or ECibN (g) were expressed, no biotinylation was observed. To facilitate an unbiased visual comparison of fluorescence intensities in ( b - g ), the panels in each column display identical minimum and maximum intensity. (h) Fluorescent intensity of Sta normalized to ECibN intensity to account for variation in expression levels in different cells shows that biotinylation is detected only in the presence of light and biotin. Normalized fluorescence intensities are tabulated in table S5 while corresponding p-values are tabulated in table S7. (i) Average Pearson’s coefficient shows ECibN and CryC colocalize in the presence of light, while Sta colocalizes to ECibN only in the presence of light and biotin. Average Pearson’s coefficient values are tabulated in table S6 while corresponding p-values are tabulated in table S8. All scale bars 10 μm. Errors: s.e. n= # of cells.

Article Snippet: CibN was generated by restriction digesting a CIB-pmEGFP plasmid (Addgene plasmid # 28240, ( )) at AgeI and NheI sites and fusing PCR amplified CIB1 and spTN (Addgene plasmid # 153002, ( )) using Gibson assembly.

Techniques: Incubation, Staining, Construct, Membrane, Comparison, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions

doi: 10.1101/2022.10.22.513249

Figure Lengend Snippet: (a) Western blot of CibN and CryC expressed in HEK293T cells, incubated with 100 μM biotin, stained for biotinylation with Sta-HRP. Cells exposed to blue light for an hour on a 10 min on/off cycle show a greater Sta-HRP signal, compared to cells kept in dark. (b-d) ECibN and CryC were coexpressed in MDCK cells with 100 μM added biotin, exposed to blue light for four hours on a 10 min on/off cycle, and stained for biotinylation, Ecad, and β-catenin. Light-exposed cells show greater light-dependent biotinylation determined by Sta-HRP compared to those kept in the dark (b), and demonstrate biotinylation of ECibN and endogenous Ecad (EcadWT) (c), and β-catenin, an adaptor protein known to interact with Ecad cytoplasmic region (d). (e) MS data for HEK293T cells show more biotinylated proteins for light-exposed cells (cyan) compared to cells kept in dark (magenta), with CIB1 and GFP (circled) being among the top-ranked in the data. (f) Principal Component Analysis (PCA) shows similar variation for all replicates of the same condition (R1, R2, R3), with light-exposed cells (cyan) calculated to be significantly different from the control cells kept in the dark (magenta) with 100 μM added biotin.

Article Snippet: CibN was generated by restriction digesting a CIB-pmEGFP plasmid (Addgene plasmid # 28240, ( )) at AgeI and NheI sites and fusing PCR amplified CIB1 and spTN (Addgene plasmid # 153002, ( )) using Gibson assembly.

Techniques: Western Blot, Incubation, Staining, Control