Sub Cellular Imaging of Proteasomal Subunits in Cultured Cells

Proteasomes are multi-subunit enzyme complexes responsible for degrading the majority of excess, misfolded, or damaged proteins in cells. Up to 30% of all newly synthesized proteins in healthy cells are misfolded and immediately sent for degradation. Impaired proteasome function causes undegraded protein accumulation, leading to cell pathology, and causes several known diseases. Classic biochemical techniques used to study proteasomal functions have been limited to measuring mean protein degradation rates. To date, the field has been unable to determine which proteasomal configurations or in what cellular locations the most protein degradation occurs in. Proteasomal machinery can be between 12-45 nm in size. Our goal is to visualize the individual proteasome configurations and their locations in control and diseased states. Using a super resolution imaging technique called Stimulated Emission Depletion (STED) the proteasomal structures, their locations and degradative activities can be visualized and measured. STED enhances the resolution of conventional microscopy by using a depletion laser that allows structures from 10-30 nm to be imaged using fluorescence. Currently proteasomal subunits are being cloned and fused with fluorescent reporter proteins to label different proteasome machinery for STED imaging. By understanding the altered proteasomal degradation dynamics that underlie disease new therapeutics can be developed