Structural characterization of toxic protein aggregates in situ
Protein aggregates are often found in neuronal cells of patients suffering from neurodegenerative diseases such as Alzheimer’s or Parkinson’s. However, little is known about the morphological characteristics of these aggregates in their native environment. Our group seeks to understand the detailed structure and cellular interactions of protein aggregates within optimally preserved cells to gain insights into their mechanisms of toxicity.
To that end we employ cryo-electron tomography (cryo-ET), a technique that allows 3D visualization of the cell interior at molecular resolution. In first place, eukaryotic cells expressing toxic aggregating proteins relevant to neurodegenerative disease (e.g. Huntingtin, Tau …) are vitrified on EM grids. This is achieved by quickly plunging the cells into a fluid cooled at liquid nitrogen temperature (-196 °C) (Figure 1A, left). Once the samples are frozen, they are kept at this temperature for all subsequent steps. Second, since eukaryotic cells are usually too thick for direct imaging at the Transmission Electron Microscope (TEM), the cells are thinned down to ~300 nm thick slices (“lamellas”) using a cryo-Focused Ion Beam (FIB) (Figure 1A, middle). If necessary, this step can be guided by correlative light/electron microscopy. Finally, lamellas are imaged in 3D using a cryo-TEM (Figure 1A, right).
Figure 1: Cryo-electron tomography workflow and Cryo-ET of a HeLa cell containing a Huntingtin aggregate. A large Huntingtin aggregate (Htt), ice crystal (IC), and the endoplasmic reticulum (ER) are indicated.[less]
Figure 1: Cryo-electron tomography workflow and Cryo-ET of a HeLa cell containing a Huntingtin aggregate. A large Huntingtin aggregate (Htt), ice crystal (IC), and the endoplasmic reticulum (ER) are indicated.
Figure 1B and C show an example of this workflow. A human HeLa cell expressing Huntingtin-exon1-GFP was plunge-frozen and targeted for FIB milling. The resulting lamella containing a large Huntingtin aggregate (Figure 1B, oval marked by Htt) was imaged in the cryo-TEM. 3D data sets were recorded in regions of interest (Figure 1C and square in Figure 1B). Figure 1C shows the edge of the Huntingtin aggregate (top) and how the fibrils can be traced using automated algorithms (bottom).