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Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly
摘要： The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that increasing surface area of the expanding aggresome increases the rate of accretion due to diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.
关键词： molecular modelling,protein aggregation,molecular imaging,passive transport,amyloid protein,aggresome formation,transport,live cell SIM,protein misfolding,molecular dynamics
更新于2025-09-23 15:21:21
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Label-free imaging of amyloid plaques in Alzheimer’s disease with stimulated Raman scattering microscopy
摘要： One of the key pathological features of Alzheimer’s disease (AD) is the existence of extracellular deposition of amyloid plaques formed with misfolded amyloid-β (Aβ). The conformational change of proteins leads to enriched contents of β sheets, resulting in remarkable changes of vibrational spectra, especially the spectral shifts of the amide I mode. Here, we applied stimulated Raman scattering (SRS) microscopy to image amyloid plaques in the brain tissue of an AD mouse model. We have demonstrated the capability of SRS microscopy as a rapid, label-free imaging modality to differentiate misfolded from normal proteins based on the blue shift (~10 cm?1) of amide I SRS spectra. Furthermore, SRS imaging of Aβ plaques was verified by antibody staining of frozen thin sections and fluorescence imaging of fresh tissues. Our method may provide a new approach for studies of AD pathology, as well as other neurodegenerative diseases associated with protein misfolding.
关键词： amyloid plaques,protein misfolding,Alzheimer’s disease,stimulated Raman scattering microscopy,label-free imaging
更新于2025-09-09 09:28:46