Publications


 


Publications

2016 2005

2016
Apetri MM, Harkes R, Subramaniam V, Canters GW, Schmidt T, Aartsma TJ (2016) Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells., PLoS ONE, 11, e0153020.
[Abstract] [DOI] [pdf]
Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.

2005
Schmauder, R, Librizzi, F, Canters, GW, Schmidt, T, Aartsma, TJ (2005) The oxidation state of a protein observed molecule-by-molecule, ChemPhysChem, 6, 1381-1386.
[DOI]
Schmauder, R, Alagaratnam, S, Chan, C, Schmidt, T, Canters, GW, Aartsma, TJ (2005) Sensitive detection of the redox state of copper proteins using, JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY, 10, 683-687.
[Abstract] [DOI] [pdf]
The blue copper protein azurin from Pseudo- monas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.