Fig. 2: Fluorescent detection of nascent lamin dimers.
From: Co-translational ribosome pairing enables native assembly of misfolding-prone subunits
A Approach for visual verification of N-terminal co-localization. Two nascent chains can be linked by the FlAsH dye, which binds to a bipartite tetracysteine motif formed by two cysteines at each N-terminus, if they co-localize as in the native coiled-coil dimer structure (Fig. 1A). Bound FlAsH becomes fluorescent and is detected by scanning a confocal excitation beam (yellow beam, green arrows) along the molecular tether, while the optical tweezers laser beams (blue) trap the beads. B Corresponding data. Bottom: detected fluorescence scans in time, showing bead movements in stretch-relax cycles and the FlAsH fluorescence signal between the two beads (green arrowhead). The FlAsH signal is only observed when the tether is under tension and hence stably in focus. RNC pair construct: LMNA31–476. Top: corresponding measured force acting on the beads and tether. Blue dashed line: sudden drop in force indicates unfolding, while FlAsH keeps the N-termini connected (A). Red dashed line: tether rupture, likely by dissociation of one of the DNA handles from the bead surface. Source data are provided as a Source Data file. RNC cartoon from Bertolini et al.16. Reprinted with permission from AAAS.
