Fig. 1.

A mosaic transfection model for quantifying nuclear protein propagation. (A) RFPs of varying molecular weight (RFP-reporter) were fused with 3× SV40 cNLS in a tetracycline-inducible vector. (B) Primary mouse myoblasts were transfected with an RFP reporter plasmid at 1 h after seeding, resulting in a sparse mosaic transfection (see also SI Appendix, Fig. S1). Myoblasts were then differentiated for 2 d without doxycycline. Following myotube fusion, doxycycline was added to the culture medium for 24 h to induce expression of the RFP-cNLS fusion protein, and the RFP distributions within individual myotubes were quantified by epifluorescence microscopy. (C) Time-lapse microscopy of a Hoechst-stained myotube following induction with doxycycline showed that the myotube contained a single transfected myonucleus expressing the DsRed-cNLS fusion protein (see also Movies S1 to S6). Initially, the RFP-cNLS fusion protein was imported into the transfected myonucleus (yellow arrow), but after 12 h, the reporter protein was also imported into neighboring myonuclei (white arrows). This process continued for 24 h after doxycycline induction, resulting in a gradient of myonuclear RFP fluorescence, which we refer to as the propagation profile for that protein. (Scale bar: 50 µm.)