Profiling of Endocytosis Pathways
In order to confirm the endocytic pathway taken by α-synuclein fibrils, I did a thorough antibody profiling of compartments that colocalized with fibrils during their internalization. Here, I tested whether α-synuclein fibrils traffic through early endosomes. I added fluorescently labeled fibrils to cells and stained for EEA1, an early endosome marker. Across multiple time points, the fibrils appeared as puncta inside cells but showed no overlap with EEA1, indicating they bypass early endosomes.
I next compared fibril localization with different endocytic markers. The fibrils did not colocalize with Rab4, Rab5, or Rab8 (early/recycling endosomes), but instead overlapped with Rab9 and LAMP1, markers of late endosomes and lysosomes. This showed that fibrils are rapidly targeted to the degradative lysosomal pathway rather than recycling routes.