A mutant form of the ataxin-1 protein with an expanded polyglutamine (polyQ) tract is the underlying cause of the inherited neurodegenerative disease spinocerebellar ataxia 1 (SCA1). In probing the biophysical features of the nuclear bodies (NBs) formed by polyQ-ataxin-1, we defined ataxin-1 NBs as spherical liquid protein/RNA droplets capable of rapid fusion. We observed dynamic exchange of the ataxin-1 protein into these NBs; notably, cell exposure to a pro-oxidant stress could trigger a transition to slower ataxin-1 exchange, typical of a hydrogel state, which no longer showed the same dependence on RNA or sensitivity to 1,6-hexanediol. Furthermore, we could alter ataxin-1 exchange dynamics either through modulating intracellular ATP levels, RNA helicase inhibition, or siRNA-mediated depletion of select RNA helicases. Collectively, these findings reveal the tunable dynamics of the liquid RNA/protein droplets formed by polyQ-ataxin-1.Within eukaryotic cells, complex biochemical reactions are facilitated by the concentration and restriction of key enzymes, substrates and regulators within well-defined membrane-bound organelles. In addition, numerous membrane-less intracellular compartments are important for a range of essential activities in both the cytoplasm (e.g. stress granules storing RNA molecules from stalled translation in response to environmental stresses 1 ) and the nucleus (e.g. nucleoli functioning in the biogenesis of ribosome subunits 2 , Cajal bodies in the processing and modification of short non-coding RNAs 3 , or promyelocytic leukemia (PML) bodies acting as sumoylation factories in response to interferon and oxidative stress 4 ). The formation of many of these membrane-less organelles is now understood to proceed via a phase separation process of specific constituent proteins, RNA and/or DNA molecules 5 . Thus, after a certain critical concentration threshold is exceeded, molecular assemblies of these constituents are formed with liquid-like behaviors that include fusing ability, viscous fluid dynamics, and high exchange rates with their surroundings in the nucleoplasm or cytoplasm 6-10 . This process of protein phase separation is now viewed as an essential mechanism for efficient compartmentalization that can be rapidly responsive to environmental challenges or intracellular changes 11,12 .Proteins that can undergo phase separation usually contain sequences conforming to either a low complexity region (LCR) or prion-like domain (PrLD) 8,11,13 ; these are protein domains typically with low amino acid diversity and little conformational heterogeneity 5,11 . These disordered structural characteristics can also contribute to an additional change known as protein phase transition, in which liquid-like condensates continue to become less dynamic and so form a more viscoelastic hydrogel or solid-like fibrous aggregates 12,14 . Many factors can promote the phase transition process such as time, pH, and altered amino acid sequences arising from gene mutations 8,9,15,16 . Thus, sequences typically co...

The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although known to form distinctive intranuclear bodies, the cellular pathways and processes that polyQ-ataxin-1 influences remain poorly understood. Here we identify the direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells, pathways analyses indicating a significant enrichment of essential nuclear transporters, pointing to disruptions in nuclear transport processes in the presence of elevated levels of ataxin-1. Our direct assessments of nuclear transporters and their cargoes confirm these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Analogous changes in importin-β1, nucleoporin 98 and nucleoporin 62 nuclear rim staining are observed in Purkinje cells of ATXN1[82Q] mice. The results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQ-ataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.

Ataxin-1 mutation, arising from a polyglutamine (polyQ) tract expansion, is the underlying genetic cause of the late-onset neurodegenerative disease Spinocerebellar ataxia type 1 (SCA1). To identify protein partners of polyQ-ataxin-1 in neuronal cells under control or stress conditions, here we report our complementary proteomics strategies of proximity-dependent biotin identification (BioID) and affinity purification (via GFP-Trap pulldown) in Neuro-2a cells expressing epitope-tagged forms of ataxin-1[85Q]. These approaches allowed our enrichment of proximal proteins and interacting partners, respectively, with the subsequent protein identification performed by liquid chromatography-MS/MS. Background proteins, not dependent on the presence of the polyQ-ataxin-1 protein, were additionally defined by their endogenous biotinylation (for the BioID protocol) or by their non-specific interaction with GFP only (in the GFP-Trap protocol). All datasets were generated from biological replicates. Following the removal of the identified background proteins from the acquired protein lists, our experimental design has captured a comprehensive polyQ-ataxin-1 proximal and direct protein partners under normal and stress conditions. Data are available via ProteomeXchange, with identifier PXD010352.

words)The expanded polyglutamine (polyQ) tract form of ataxin-1 drives disease progression in spinocerebellar ataxia type 1 (SCA1). Although polyQ-ataxin-1 is known to form distinctive intranuclear bodies, the cellular pathways and functions it influences remain poorly understood. Here, we identify direct and proximal partners constituting the interactome of ataxin-1[85Q] in Neuro-2a cells. Pathways analyses indicate a significant enrichment of essential nuclear transporters in the interactome, pointing to disruptions in nuclear transport processes in the presence of polyQ-ataxin-1. Our direct assessments of nuclear transporters and their cargoes reinforce these observations, revealing disrupted trafficking often with relocalisation of transporters and/or cargoes to ataxin-1[85Q] nuclear bodies. Strikingly, the nucleoporin Nup98, dependent on its GLFG repeats, is recruited into polyQ-ataxin-1 nuclear bodies. Our results highlight a disruption of multiple essential nuclear protein trafficking pathways by polyQataxin-1, a key contribution to furthering understanding of pathogenic mechanisms initiated by polyQ tract proteins.