A method to rapidly create protein aggregates in living cells

Abstract

The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.

Figure 1. Development of AgDD to rapidly create protein aggregates in living cells.

(a) Schematic representation of inducing aggregated proteins inside cells using an AgDD. (b) Images of representative HEK cells stably expressing AgDD before and after S1 washout (WO) for 150 min. Scale bar, 10 μm. (c) Time-lapse images of representative HEK cells stably expressing AgDD. Top: cells cultured in media with S1 throughout the experiment; middle: cells cultured in media with S1 withdrawn at 0 h and bottom: cells cultured in media with S1 withdrawn from 0–1 h then readministered from 1–8 h. Scale bars, 20 μm. (d) Early time-lapse images of representative HEK cells stably expressing AgDD following S1 withdrawal. Scale bar, 20 μm.

Figure 2. AgDD aggregates have similar characteristics as filamentous aggregates.

(a) TEM image of representative HEK cells stably expressing AgDD after S1 washout for 60 min. Scale bar, 5 μm. (b) Magnified TEM image of white box inset in a. Scale bar, 1 μm. (c) Immunofluorescence images of representative cells stably expressing AgDD-GFP before and after S1 removal for the indicated times. Left: AgDD; middle-left: p62; middle-right: Hoechst; right: merge. Scale bars, 10 μm. Texas-Red was used to label the p62 antibody.

Figure 3. Applications of AgDD.

(a) HEK293 cells expressing AgDD and HEK293 cells expressing mCherry were mixed and co-cultured in media containing S1. S1 was withdrawn for the indicated times, and cell populations were quantified using flow cytometry. Replicates of n=3. Error bar is Standard Deviation (STD). (b) Time-lapse images of representative HEK293 cells stably expressing Nuclear AgDD. Top: cells cultured in media with S1 throughout the experiment; bottom: cells cultured in media with S1 withdrawn at 0 h. Scale bars, 5 μm. (c) Early time-lapse images of representative HEK293 cells stably expressing nuclear AgDD following S1 removal. Scale bar, 5 μm. (d–g) Images of representative nematodes expressing AgDD in intestinal cells. Top left: no S1; top right: 50 μM S1, bottom left: S1 removed for 2 h, bottom right: S1 removed for 6 h. Scale bar, 50 μm.