PMC

Confocal laser scanning microscopy composite images of Yn-GC-MD and Yn-GD-UHC-MD cells.

Time course of CMC hydrolysis using yeast cells displaying CelA via different assembly methods ().

UHC-expressing E. coli BL21 cells showed green fluorescence when incubated with 10 μM ThT. No fluorescence was noted when ThT was incubated with E. coli ells expressing CelA, which were used as controls.

Flow cytometry analysis of yeast cells with different molecular assembly display treatments. Note that significantly higher numbers of cells display an elevated level of mCherry fluorescence when UHC fibrils are used (i.e., Yn-GD-UHC-MD).

Probing protein complex formation on the surface of yeasts displaying GFP-nanobody (Yn) via epifluorescence microscopy. Yn+MD, Yn incubated with MD; Yn-GD+MD, Yn sequentially incubated with GD and MD; Yn+UHC+MD, Yn sequentially incubated with UHC and MD; Yn-GC-MD, Yn sequentially incubated with GC and MD; Yn-GD-UHC-MD, Yn sequentially incubated with GD, UHC fibrils, and MD; Yn-GD-UHC-MD/GD, Yn sequentially incubated with GD, UHC fibrils, and a mixture of MD and GD.

Proposed schematics of the protein complexes assembled on the cell surface of Yn-GD-UHC-MD yeast (a) and Yn-GC-MD yeast (b). I, AGN (yeast Aga2p fused with GFP nanobody); II, GD (GFP-dockerin); III, UHC (Ure2p^1-80 fused with HD and cohesin); IV, MD (mCherry-dockerin); V, GC (GFP-cohesin).

Characterization of UHC fibrils. (a) ThT fluorescence emission spectrum of UHC fibrils incubated with ThT and excited at 450 nm. (b) Kinetics of UHC fibril formation monitored using ThT binding. (c and d) Influence of BSA on the formation of UHC fibrils. UHC (20 μM) was incubated in TBS buffer without (c) or with (d) 0.5% BSA at 22°C with shaking and then stained with ThT before being viewed with a fluorescence microscope. Scale bar, 10 μm. (e) Formation of amyloid-like fibrils by the UHC proteins, as revealed by AFM (scan area, 5 by 5 μm²). AU, arbitrary units.