Jan 13, 2025
Quantification of endolysosomal leak of alpha-synuclein fibrils by fluorescence complementation
- Cole Sitron1,
- F Ulrich Hartl1
- 1Max Planck Institute of Biochemistry
Protocol Citation: Cole Sitron, F Ulrich Hartl 2025. Quantification of endolysosomal leak of alpha-synuclein fibrils by fluorescence complementation. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71dr8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 103707
Keywords: ASAPCRN, synuclein fibrils by fluorescence complementation, synuclein fibril, synuclein, quantification of endolysosomal leak, fluorescence complementation, endocytosed fibril, endolysosomal leak, leakage of endocytosed alpha, fibril, fluorescence, flow cytometry, nuclear export sequence in cell, endocytosed alpha, fluorescence microscopy
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000282
Aligning Science Across Parkinson’s
Grant ID: ASAP- 024268
Abstract
This technique uses a bipartite split mNeonGreen system to detect leakage of endocytosed alpha-synuclein fibrils into the cytoplasm. The 11th beta strand of mNeonGreen-3K is fused to the alpha-synuclein preformed fibrils (PFFs) and the conjugate 1-10th beta strands are expressed with a nuclear export sequence in cells. If the endocytosed fibrils leak into the cytoplasm, the mNeonGreen-3K fragments will complement, generating a fluorescent signal that can be detected by flow cytometry or fluorescence microscopy.
Materials
• A cell line stably expressing NES-mNeonGreen-3K-1-10-IRES-mKate2
• 5mg/ml alpha-synuclein A53T PFFs tagged with mNeonGreen-3K-11 (generated exactly as in dx.doi.org/10.17504/protocols.io.btynnpve )
• Appropriate cell culture medium
• OptiMEM (Thermo Fisher Scientific cat. no. 31985062)Opti-MEM Gibco - Thermo Fisher ScientificCatalog #31985062
• Phosphate-buffered saline (PBS) (Thermo Fisher Scientific cat. no. 20012068)PBS, pH 7.2Thermo FisherCatalog #20012068
• Dulbecco's Phosphate-Buffered Saline (DPBS) (Thermo Fisher Scientific cat. no. 14190144)Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo Fisher ScientificCatalog #14190144
• Poly-D-Lysine 0.1 mg/ml (Thermo Fisher Scientific cat. no. A3890401)Poly-D-LysineThermo Fisher ScientificCatalog #A3890401
• BioRuptor Plus sonicator (Diagenode cat. no. B01020001)Bioruptor Plus sonication systemDiagenodeCatalog #B01020001 (or equivalent)
• 12-well cell culture plates (e.g. Falcon cat. no. 353043)Falcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WrCorningCatalog #353043
Troubleshooting
Methods
30m
Dilute Poly-D-Lysine 1:2 in DPBS.
Coat cell culture plates with diluted Poly-D-Lysine Overnight with rocking.
Wash the wells three times with MilliQ water.
Remove the MilliQ water and air dry for 00:30:00 . Plates can now be stored at 4 °C for two weeks.
30m
Plate NES-mNeonGreen-3K-1-10-IRES-mKate2 75000 cells per well into coated plates.
Thaw PFFs and dilute 1:20 into a tube containing PBS.
Note
The final volume must be between 100 µL and 300 µL to ensure proper sonication.
Sonicate the PFFs in the Bioruptor Plus on high for 25 cycles of 00:00:05 on and 00:00:05 off at 4 °C .
10s
Make a master mix of OptiMEM/PFF or OptiMEM/PBS, with a ratio of 100 µL OptiMEM to 40 µL diluted PFFs or PBS.
Add 140 µL of OptiMEM/PFF or OptiMEM/PBS dropwise to each well.
Allow the plate to incubate for two days before downstream analysis by flow cytometry or microscopy.
Note
If analyzing cells by flow cytometry, it is critical to prepare a PBS control for each condition. mNeonGreen-positive cells can be identified by first drawing a gate above the PBS-treated population on a BL1-A vs BL2-A plot. This plot allows for autofluorescence correction, as autofluorescence should alter both the BL1-A and BL2-A channel, while mNeonGreen fluorescence should only increase the BL1-A channel signal. After drawing the gate based on the PBS-treated control, one can calculate the proportion of cells in the PFF-treated sample that lay within the gate.
Protocol references
Citations
- Zhou S, Feng S, Brown D, Huang B. Improved yellow-green split fluorescent proteins for protein labeling and signal amplification. PLoS One. 2020 Nov 23;15(11):e0242592. doi: https://doi.org/10.1371/journal.pone.0242592. PMID: 33227014; PMCID: PMC7682878.