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In JoVE (1)
Other Publications (2)
Articles by Yasmin M. Ramdzan in JoVE
ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells
Sevgi Irtegun, Yasmin M. Ramdzan, Terrence D. Mulhern, Danny M. Hatters
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute
The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.
Other articles by Yasmin M. Ramdzan on PubMed
Chemistry & Biology. Apr, 2010 | Pubmed ID: 20416508
Proteins prone to misfolding form large macroscopic deposits in many neurodegenerative diseases. Yet the in situ aggregation kinetics remains poorly understood because of an inability to demarcate precursor oligomers from monomers. We developed a strategy for mapping the localization of soluble oligomers and monomers directly in live cells. Sensors for mutant huntingtin, which forms aggregates in Huntington's disease, were made by introducing a tetracysteine motif into huntingtin that becomes occluded from binding biarsenical fluorophores in oligomers, but not monomers. Up to 70% of the diffusely distributed huntingtin molecules appeared as submicroscopic oligomers in individual neuroblastoma cells expressing mutant huntingtin. We anticipate the sensors to enable insight into cellular mechanisms mediated by oligomers and monomers and for the approach to be adaptable more generally in the study of protein self-association.
Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool
The Journal of Biological Chemistry. Jul, 2010 | Pubmed ID: 20444706
Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100-6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s(20,w) = 140 S) and IBs (mode s(20,w) = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.