Visualizing protein aggregation

In brain disease, certain proteins transition from a soluble to insoluble and dysfunctional state, we call this protein aggregation. We still do not fully understand what causes proteins in the brain to aggregate and whether this is a cause or an effect of disease. To identify important factors that regulate protein aggregation we  use a combination of biophysical studies and cell biology.

A) Protein aggregation in the test tube

An amazing feature of proteins that aggregate in the brain is that when they are isolated from the cell they have an intrinsic propensity to self-aggregate. An example of this is a phenomenon called liquid-liquid phase separation (LLPS). LLPS is a biophysical event where protein condenses into concentrated protein-rich liquid droplets (see here for a description). Using the formation of liquid droplets as a read out we can add substrates such as poly(ADP-ribose) (PAR) to the protein and examine the resulting effect (see panel A above). These experiments help us understand the regions of the protein critical for this event as well as the external substrates that may alter how the protein behaves.

B) Protein aggregation in the cell

Exposing cells to an external stress can lead to a regulated protein-aggregation response, and we can watch this happen using microscopy techniques (see panel B). For example, in the cytoplasm, proteins and RNA complexes that are poised to initiate protein translation, condense into membraneless organelles called stress granules (see here for a description). Stress granules are thought to form by a liquid-liquid phase separation event (see above and panel A). Many of the proteins that aggregate in dementia and motor neuron disease can be found in stress granules. We use immunofluorescence techniques in mammalian cells and in neuronal cultures to understand the relationship between stress granules, the disease protein and the disease process. Using this cellular assay I initiate drug screens as a means to translate what we uncover into potential therapeutic strategies.