Endocytosis
Live-Cell Uptake Imaging
I applied real-time confocal and super-resolution microscopy to capture the earliest events in α-synuclein fibril internalization. Remarkably, fibrils bypassed early endosomes and reached lysosomes within two minutes of exposure. These findings established a rapid, clathrin-independent uptake pathway distinct from classical endocytosis.
Confocal imaging showing rapid uptake of fluorescently labeled α-synuclein fibrils by SH-SY5Y cells, with fibrils bypassing early endosomes and localizing to lysosomes within minutes of exposure.
Following endocytic entry using EM
To capture these images, we combined nanogold labeling of α-synuclein fibrils with transmission electron microscopy (TEM). This approach enabled high-resolution visualization of fibril localization at the plasma membrane, within macropinosomes, and in multivesicular bodies, providing ultrastructural evidence for their uptake and trafficking pathways.
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(i) Nanogold-labeled α-synuclein fibrils are seen at the plasma membrane, closely associated with large ruffling regions.
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(ii) Ruffles begin to fold around fibrils, forming circular structures consistent with the initiation of macropinocytosis.
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(iii) Fibrils are enclosed within newly formed macropinosomes, confirming their internalization via this pathway.
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(iv) Some fibrils appear in multivesicular bodies (MVBs) shortly after uptake, suggesting rapid sorting toward vesicles involved in exosomal release.
Macropinocytosis Inhibition
Confocal imaging of human astrocytes treated with α-synuclein fibrils shows that pharmacological inhibitors of macropinocytosis strongly block fibril uptake. EIPA, which disrupts Na⁺/H⁺ exchange, and Latrunculin B, which prevents actin polymerization, both significantly reduced internalization of fibrils. Importantly, uptake of control cargo such as EGF remained unaffected, confirming that the observed effect was specific to the macropinocytic pathway
SARS-CoV-2 Internalization Study
This study uncovered how SARS-CoV-2 gains entry into human cells. I found that the viral spike protein attaches to ACE2 on the cell surface and is quickly pulled inside through clathrin-mediated endocytosis, a major cellular uptake pathway. When this pathway was blocked, the virus could no longer get in, showing that hijacking endocytosis is essential for infection.
Pseudotyped SARS-CoV-2 Lentivirus
To test whether SARS-CoV-2 entry depends on clathrin-mediated endocytosis, we used a pseudovirus system. Lentiviral particles pseudotyped with the SARS-CoV-2 spike protein were applied to HEK-293T cells stably expressing ACE2 following plasmid transfection. Infection efficiency was measured by GFP reporter expression. In parallel, cells were treated with siRNA targeting clathrin heavy chain (CHC) to disrupt clathrin-mediated endocytosis. Compared to control siRNA, CHC knockdown significantly reduced pseudovirus infectivity, demonstrating that clathrin-dependent pathways are essential for viral entry.