Figure 6. Application of the optimized... | Rockefeller University Press

Figure 6.

$Application of the optimized optogenetic toolbox to populations of cells.(A) Constructs: use of a P2A self-cleaving peptide enables expression of iLID-mCherry-RAB11 with SSPB-Kin14-VIb or opto-kinesin-SSPB at equimolar levels from a single construct. (B) Immunoblot (IB) of whole-cell lysates from U2OS cells expressing the indicated proteins. Dotted lines indicate the excision of lanes. Exp. MW, expected molecular weight. (C–F) Fixed-cell imaging (C) and quantification (D–F) of iLID-mCherry-RAB11 and opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm−2). (D) Quantification showing the NPF of RAB11 intensity. (E) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and iLID-mCherry-RAB11 when expressed from a single plasmid (magenta) as in A or using two separate constructs for motor and cargo (gray). (F) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and the peripheral fraction of RAB11 per cell in the dark (gray) and after illumination (blue). (G and H) Fixed-cell imaging (G) and quantification (H) of iLID-mCherry-RAB11 and SSPB(micro) in COS-7 from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm−2). Graph in H shows the normalized perinuclear fraction of RAB11 intensity (mean RAB11 MTOC intensity/mean cellular RAB11 intensity). (I) Live-cell imaging of iLID-mCherry-RAB11 in HeLa Flp-in cells treated with doxycycline to induce the equimolar expression of SSPB-GCN4-mVenus–ppKin14-VIb and iLID-mCherry-RAB11 (top; see also Video 9) or of KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) and iLID-mCherry-RAB11 (bottom; see also Video 10) before or during illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset. Data were from at least three experiments. Scale bars are 10 µm.$

Application of the optimized optogenetic toolbox to populations of cells. (A) Constructs: use of a P2A self-cleaving peptide enables expression of iLID-mCherry-RAB11 with SSPB-Kin14-VIb or opto-kinesin-SSPB at equimolar levels from a single construct. (B) Immunoblot (IB) of whole-cell lysates from U2OS cells expressing the indicated proteins. Dotted lines indicate the excision of lanes. Exp. MW, expected molecular weight. (C–F) Fixed-cell imaging (C) and quantification (D–F) of iLID-mCherry-RAB11 and opto-kinesin KIF1A(1–365)-VVDfast-GFP-SSPB(micro) from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm⁻²). (D) Quantification showing the NPF of RAB11 intensity. (E) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and iLID-mCherry-RAB11 when expressed from a single plasmid (magenta) as in A or using two separate constructs for motor and cargo (gray). (F) Graph showing normalized cellular expression levels of KIF1A(1–365)-VVDfast-GFP-SSPB(micro) and the peripheral fraction of RAB11 per cell in the dark (gray) and after illumination (blue). (G and H) Fixed-cell imaging (G) and quantification (H) of iLID-mCherry-RAB11 and SSPB(micro) in COS-7 from a single plasmid in COS-7 cells in the dark or after illumination with blue light (100 µW cm⁻²). Graph in H shows the normalized perinuclear fraction of RAB11 intensity (mean RAB11 MTOC intensity/mean cellular RAB11 intensity). (I) Live-cell imaging of iLID-mCherry-RAB11 in HeLa Flp-in cells treated with doxycycline to induce the equimolar expression of SSPB-GCN4-mVenus–ppKin14-VIb and iLID-mCherry-RAB11 (top; see also Video 9) or of KIF1A(1–365)-VVDfast-mVenus-SSPB(micro) and iLID-mCherry-RAB11 (bottom; see also Video 10) before or during illumination. Dots represent one cell and bars indicate mean and 95% confidence intervals. Dotted lines represent the mean and the mean ± two times the SD of control cells. Numbers show the percentage of responsive cells for each dataset. Data were from at least three experiments. Scale bars are 10 µm.