Amyloids, aggregates and Nucleolar activity in Xenopus development

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Project Summary: Many cellular processes critical for normal development have devastating consequences when they are not controlled. Not too long ago, protein aggregation was largely equated with pathogenesis. More recently we know that proteins aggregate to mediate stress responsiveness, cell division, long-term memory and to self-assemble into critical non-membrane bound particles in the nucleus and cytosol. If fact, at least 5% of known proteins have the composition that would allow them to form aggregates. Lack of proper cellular control of protein aggregation leads to amyloid formation, which unregulated, is part of the pathogenesis that accompanies devastating diseases like Alzheimer’s and Huntington’s disease and common debilitating conditions like cataracts. The physical nature of aggregates may be that of phase separated liquid droplets, gels, disordered aggregates and the ordered aggregates characteristic of amyloid. Aggregates serve to greatly increase the local concentration of the aggregating protein, and are one part of an equilibrium between different structural states. Consequently, the ability and rate of proteins joining or leaving an aggregate is an important feature that can be regulated, and if compromised leads to aggregate based pathology. There is a gap in understanding which proteins participate in aggregate formation and how aggregation is regulated in vivo. We propose that an ideal system to begin to bridge the gap would be the oocytes of the frog Xenopus laevis. We and others have shown that aggregates accumulate during oogenesis in the cytosol and in nucleus. The nuclear particles responsible for RNA transcription and processing by all three RNA polymerases self-assemble into aggregates, generally with RNA as a co-aggregate. The focus of this proposal is on nucleolus, a prominent non-membrane bound particle responsible for synthesis and processing of ribosomal RNA, the manufacturing of ribosomes, helping to mediate cellular responses to stress and DNA damage, and appropriately responding to cellular need for new translational capacity. We use microscopy, immunohistochemistry, biochemical fractionation, protein depletion, proteomic and transcriptomic analysis to understand the accumulation, function and stability of the aggregates that establish nucleolar form and function. We take advantage of the unique properties of Xenopus oocyte nuclei, including size, abundant protein and RNA content and ease of manipulation to perform our studies. The findings will impact our understanding of how cells maintain physiologically normal equilibrium between native and aggregating forms of proteins.