Hinde, E. — Page 2 | bioRxiv Science

bioRxiv2020

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

Mutations that cause Huntingtons Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntingtons disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and examined whether ribosome quality control machinery previously implicated in stalled ribosomes were involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation.

cell biology↗

bioRxiv2018

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organised at a double strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image correlation spectroscopy (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.\n\nSIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

biophysics↗