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Different effects of alpha-Synuclein mutants on lipid binding and aggregation detected by single molecule fluorescence spectroscopy and ThT fluorescence-based measurements
摘要： Six alpha-Synuclein point mutations are currently known to be associated with familial parkinsonism: A30P, E46K, H50Q, G51D, A53E and A53T. We performed a comprehensive in vitro analysis to study the impact of all aSyn mutations on lipid binding and aggregation behavior. Markedly reduced lipid binding of A30P, moderately attenuated binding of G51D and only very slightly reduced binding for the other mutants were observed. A30P was particularly prone to form metal ion induced oligomers, whereas A53T exhibited only weak tendencies to form oligomers. In turn, fibril formation occurred rapidly in H50Q, G51D and A53T, but only slowly in A30P, suggesting mutants prone to form oligomers tend to form fibrils to a lesser extent. This was supported by the observation that fibril formation of wild type aSyn, A30P and A53T was impaired in the presence of ferric iron. Additionally, we found the aggregation kinetics of mixtures of A30P or A53T and wt aSyn to be determined by the faster aggregating aSyn variant. Our results implicate differential mechanisms playing a role in aSyn pathology on the molecular level. This might contribute to a better understanding of Parkinson′s disease pathogenesis and provide potential links to develop prevention strategies and disease-modifying therapy.
关键词： synucleinopathy,Alpha-synuclein (α‐Synuclein),Parkinson′s disease,protein‐lipid interaction,protein aggregation,mutant
更新于2025-09-23 15:23:52
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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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Quantification of Cellular Proteostasis in Live Cells by Fluorogenic Assay Using the AgHalo Sensor
摘要： Proper cellular proteostasis is essential to cellular ?tness and viability. Exogenous stress conditions compromise proteostasis and cause aggregation of cellular proteins. We have developed a ?uorogenic sensor (AgHalo) to quantify stress-induced proteostasis de?ciency. The AgHalo sensor uses a destabilized HaloTag variant to represent aggregation-prone cellular proteins and is equipped with a series of ?uorogenic probes that exhibit a ?uorescence increase when the sensor forms either soluble oligomers or insoluble aggregates. Herein, we present protocols that describe how the AgHalo sensor can be employed to visualize and quantify proteome stress in live cells using a direct ?uorescence read-out and visualization with a ?uorescence microplate reader and a microscope. Additionally, protocols for using the AgHalo sensor in combination with ?uorogenic probes and commercially available HaloTag probes to enable two-color imaging experiments are described. These protocols will enable use of the AgHalo sensor to visualize and quantify proteostasis in live cells, a task that is dif?cult to accomplish using previous, always-?uorescent methods.
关键词： protein aggregation,?uorescence intensity,proteostasis,?uorogenic sensor
更新于2025-09-23 15:21:21
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A discontinuous Galerkin model for fluorescence loss in photobleaching of intracellular polyglutamine protein aggregates
摘要： Background: Intracellular phase separation and aggregation of proteins with extended poly-glutamine (polyQ) stretches are hallmarks of various age-associated neurodegenerative diseases. Progress in our understanding of such processes heavily relies on quantitative fluorescence imaging of suitably tagged proteins. Fluorescence loss in photobleaching (FLIP) is particularly well-suited to study the dynamics of protein aggregation in cellular models of Chorea Huntington and other polyQ diseases, as FLIP gives access to the full spatio-temporal profile of intensity changes in the cell geometry. In contrast to other methods, also dim aggregates become visible during time evolution of fluorescence loss in cellular compartments. However, methods for computational analysis of FLIP data are sparse, and transport models for estimation of transport and diffusion parameters from experimental FLIP sequences are missing. Results: In this paper, we present a computational method for analysis of FLIP imaging experiments of intracellular polyglutamine protein aggregates also called inclusion bodies (IBs). By this method, we can determine the diffusion constant and nuclear membrane transport coefficients of polyQ proteins as well as the exchange rates between aggregates and the cytoplasm. Our method is based on a reaction-diffusion multi-compartment model defined on a mesh obtained by segmentation of the cell images from the FLIP sequence. The discontinuous Galerkin (DG) method is used for numerical implementation of our model in FEniCS, which greatly reduces the computing time. The method is applied to representative experimental FLIP sequences, and consistent estimates of all transport parameters are obtained. Conclusions: By directly estimating the transport parameters from live-cell image sequences using our new computational FLIP approach surprisingly fast exchange dynamics of mutant Huntingtin between cytoplasm and dim IBs could be revealed. This is likely relevant also for other polyQ diseases. Thus, our method allows for quantifying protein dynamics at different stages of the protein aggregation process in cellular models of neurodegeneration.
关键词： FLIP,Computational method,Multi-compartment,Rate coefficient,Discontinuous Galerkin,Calibration,Protein aggregation
更新于2025-09-04 15:30:14