In Situ Architecture and Cellular Interactions of PolyQ Inclusions

Abstract

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment

“…This is remarkably different from our recent observations on polyQ inclusions, which interact with and may disrupt the membranes of the endoplasmic reticulum and other organelles, but do not harbor substantial numbers of 26S proteasomes or other large macromolecules (Bauerlein et al, 2017). This difference is surprising as proteasomes were also reported to colocalize with polyQ aggregates (Bennett et al, 2005; Waelter et al, 2001).…”

“…To investigate the nature of these densities, neurons were co-transduced with untagged poly-GA and tagRFP-p62, which co-localizes with poly-GA aggregates (May et al, 2014; Mori et al, 2013b; Yamakawa et al, 2015; Zhang et al, 2014) and allows targeting untagged poly-GA by correlative microscopy. As for polyQ fibrils (Bauerlein et al, 2017), the decorating densities were absent from untagged poly-GA ribbons (Figure 1F), demonstrating that these additional densities require GFP for their formation and that the ribbons consisted indeed of poly-GA aggregates. Thus, poly-GA forms amyloid-like ribbons in neurons.…”

“…Unlike polyQ fibrils (Bauerlein et al, 2017), poly-GA ribbons did not visibly interact with cellular endomembranes. However, both the aggregate interior and periphery were densely populated by macromolecular complexes (Figure 1A, C, E, F).…”

“…The ribbons were twisted along their axis with a variable helical pitch, and often bifurcated and/or associated laterally with neighboring ribbons (Figure 1D bottom). This polymorphism contrasts with the uniform fibrils forming polyQ-expanded huntingtin exon 1 aggregates in mammalian cells (Bauerlein et al, 2017). Poly-GA ribbons were also more densely packed than polyQ fibrils, which occupied a lower fraction of the inclusion volume (poly-GA, ≥ 10 %; polyQ, ≤ 4 %).…”

“…The cultures were then vitrified and subsequently imaged by cryo-light microscopy to locate cellular poly-GA inclusions (Figure S1A). Correlative microscopy allowed the production of 100–200 nm-thick lamellas at the location of these aggregates using cryo-focused ion beam milling (Bauerlein et al, 2017; Rigort et al, 2012) (Figure S1B–D). Lastly, the samples were transferred to a cryo-transmission electron microscope for high resolution 3D imaging by cryo-ET (Figure S1E, F).…”