Threshold concentration and random collision determine the growth of the huntingtin inclusion from a stable core

Abstract

The processes underlying formation and growth of unfolded protein inclusions are relevant to neurodegenerative diseases but poorly characterized in living cells. In S. cerevisiae, inclusions formed by mutant huntingtin (mHtt) have some characteristics of biomolecular condensates but the physical nature and growth mechanisms of inclusion bodies remain unclear. We have probed the relationship between concentration and inclusion growth in vivo and find that growth of mHtt inclusions in living cells is triggered at a cytoplasmic threshold concentration, while reduction in cytoplasmic mHtt causes inclusions to shrink. The growth rate is consistent with incorporation of new material through collision and coalescence. A small remnant of the inclusion is relatively long-lasting, suggesting that it contains a core that is structurally distinct, and which may serve to nucleate it. These observations support a model in which aggregative particles are incorporated by random collision into a phase-separated condensate composed of a particle-rich mixture.

Threshold inclusion size triggers conversion of huntingtin to prion-like state that is reversible in newly born cells

“…10 Concordantly, inclusions shrink when cytoplasmic mHtt-GFP is reduced below a threshold concentration using auxin-mediated degradation. 22 Using the same strategy, we tested the ability of later stage inclusions to release material at low cytoplasmic mHtt- GFP concentrations by expressing mHtt-degron-GFP in cells co-expressing the plant E3 ligase, Tir1. In the presence of the synthetic auxin naphthalene acetic acid (NAA), cytoplasmic mHtt- degron-GFP dropped to background levels in many cells (Fig.…”

“…For time-lapse experiments, cells were transferred to a glass-bottom dish (Mat-Tek, Ashland, MA) as previously described. 22 Fields of cells containing one or more cells with an intermediate, clastic or large ovoid inclusion were selected and imaged once per hour for 6-10 hours. We have previously observed that the rapid turnover of mHtt-GFP prevents fully correcting mHtt-GFP images for photobleaching.…”

“…Auxin treatment and image analysis were performed as previously described. 22 In brief, cells were transferred to a glass-bottom dish and 8-12 fields containing at least one cell with a late-stage inclusion were chosen for imaging. Naphthalene acetic acid (Sigma Aldrich) was added to the medium to a final concentration of 250 mM, and z-stacks were collected every 15 minutes for 4-5 hours.…”

“…Htt-GFP and Hsp104-mCherry cytoplasmic intensity and integrated intensity inside the inclusion were measured using a script, previously described. 22 In brief, mean cytosolic intensity was measured and the inclusion was segmented using a threshold of 1.5x the mean cytoplasmic intensity in the optical section with the highest maximum intensity. The Analyze Particles function was used to measure the area and mean intensity of the inclusion.…”

Peripheral sequestration of huntingtin delays neuronal death and depends on N-terminal ubiquitination

“…Peskett et al, 2018) or yeast (e.g. Peskett et al, 2018;Aktar et al, 2019;Pei et al, 2021). Second, the neurons were followed continuously for weeks at relatively short time intervals, allowing for longitudinal measurements at the level of individual aggregates and neurons.…”