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. 2023 Mar 10;12:e84391. doi: 10.7554/eLife.84391

Figure 4. Intercellular transfer of Cas9 protein through open-ended tubular connections.

(A, B) Visualization of open-ended tubular connection structure and time-lapse imaging of the co-culture. Donor cells: HEK293T with stable overexpression of SBP-Cas9-GFP/gRNA and Myc-streptavidin-CD63-mCherry (Str-CD63/SBP-Cas9/gRNA). Acceptor cell: MDA-MB-231. Green: SBP-Cas9-GFP; red: Myc-streptavidin-CD63-mCherry; purple: CF640R WGA conjugate. Scale bar is 10 μm. The white arrows in (A) indicate the formation of an open-ended tubular connection. The white arrows in (B) indicate that the cargos including endosomal vesicles (red, middle row) and SBP-Cas9-GFP (green, bottom row) transferred through the open-ended tubular connection.

Figure 4.

Figure 4—figure supplement 1. MDA-MB-231 and HEK293T wild-type cells were observed by confocal microscopy.

Figure 4—figure supplement 1.

The cells were stained by Hoechst 33342 and CF640R WGA conjugate. Scale bar is 20 μm.
Figure 4—video 1. Intercellular transfer of Cas9 through open-ended tubular connections.
Download video file (10.2MB, mp4)
Formation of open-ended tubular connection and detection of endosomal vesicles (red) and SBP-Cas9-GFP (green) transferred from donor to acceptor followed by disruption of the connection. Donor cells: HEK293T with stable overexpression of SBP-Cas9-GFP/gRNA and Myc-streptavidin-CD63-mCherry (Str-CD63/SBP-Cas9/gRNA). Acceptor cell: MDA-MB-231. Scale bar is 10 μm. Green: SBP-Cas9-GFP; red: Myc-streptavidin-CD63-mCherry; purple: CF640R WGA conjugate.