Figure 6.

Dynamic imaging of aggresome growth. A, cartoon to demonstrate the two-color labeling protocol. Cells expressing SNAP-HDQ72 were labeled with TMR-Star at day 0 (red color) and 3 days later with 505-Star (green). Confocal images reveal two types of aggresome. In early-stage aggresomes, newly added material diffuses into core domains, leading to complete overlap of the two colors in the images. In contrast, late-stage aggresomes become increasingly impermeable (dense), and the newly adsorbed material remains spatially distinct from the core region. The merged Z-stack was obtained by cropping over a region containing the aggresome from the original confocal image stack. This region was then rendered by the 3-D viewer in Fiji, and the contrast was adjusted to highlight the extent of the aggresome. Scale bar, 5 μm. Right panel, confocal slices for agg2. B, 3-D SIM images of an impermeable aggresome. Labeling protocol as in A. Both the inner part and peripheral regions of aggregates were composed of small clusters. Scale bar, 2 μm. The cells were stained with Hoechst 33342 (blue). C, sequential labeling with alternating dyes reveals that the core region becomes increasingly impermeable over time. Scale bar, 7.5 μm. The cells were stained with Hoechst 33342 (blue).