Mutations in ubiquilin‐2 (UBQLN2), a ubiquitin‐binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that ...wild‐type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid–liquid phase separation in vitro. However, the impact of ALS‐linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry‐fused UBQLN2 with five patient‐derived ALS‐linked mutations and employ live‐cell imaging and photokinetic analysis to investigate how each of these mutations impact stress‐induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild‐type and mutant UBQLN2 condensates are generally cytoplasmic and liquid‐like. However, mutant UBQLN2 forms fewer stress‐induced UBQLN2 condensates than wild‐type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress‐induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild‐type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS‐linked mutations are both position‐ and amino acid‐dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress‐induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.
Mutations in ubiquilin‐2 (UBQLN2), a proteasomal shuttle protein, can lead to amyotrophic lateral sclerosis. We previously determined that wild‐type UBQLN2 forms stress‐ induced biomolecular condensates. In Riley et al., we demonstrate that patient‐derived ALS‐ linked mutations in UBQLN2 impact stress‐induced UBQLN2 condensate assembly and condensate material properties using live‐cell imaging and fixed cell experiments. Exogenously expressed UBQLN2 forms stress‐induced condensates independent of stress granules (unlike endogenous UBQLN2). Mutations such as P506T reduced condensate formation and impacted condensate viscoelastic properties. Disease mutations hinder the ability for UBQLN2 to undergo biomolecular condensate formation; this may be another potential pathogenic mechanism in ALS.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the ...neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells.
To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type and evx1;evx2 double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes in evx1;evx2 double mutants and wild-type siblings.
Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development, evx1;evx2 double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes are hmx2 and hmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression of skor1a and nefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulate skor1a and nefma expression in V0v interneurons by repressing Hmx2/3a expression.
This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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