18 Questions
How were dual positive cells collected in this study?
Via FACS
What is the purpose of fixing and mounting cells after collection?
To prepare them for microscopy imaging
What is indicated by the presence of MTBR-YFP inclusions inside cells expressing mCherry?
Transfer of MTBR-YFP aggregates had occurred
How did the researchers test if Tau aggregates transfer between co-cultured cells is inducible?
By exposing cells to MTBR Tau aggregates
What was the effect of treatment with aggregated species on mCherry cells?
Tau-YFP aggregation and transfer of these species
What technique was used to image the sorted dual positive cells?
Fluorescence microscopy
What antibodies were used to stain the cells in this experiment?
HA-Y11 antibody and rhodamine-conjugated anti-rabbit antibody
What were the two types of cells used in the co-culture experiments?
C17.2 cells and SV40-mCherry expressing cells
How were the cells fixed before imaging?
Cells were fixed with 4% paraformaldehyde for 7 minutes
What method was used to collect dual positive cells?
FACSAria cell sorter
What imaging technique was used in this experiment?
Confocal microscopy
What components were present in the cell lysis buffer?
10 mM Tris-HCl, 0.8 M NaCl, 1 mM EGTA, 5 mM EDTA, 10% sucrose, protease inhibitor tablet
What was the method used to harvest cells for flow cytometry?
Cells were harvested with 0.25% trypsin for 3 minutes
How many times was each experiment repeated, and how many cells were counted in each individual experiment?
Each experiment was repeated four times, and 10,000 cells were counted in each individual experiment
What was the composition of the Sarkosyl-insoluble pellets resuspension solution?
50 !l of 10 mM HEPES and 100 mM NaCl (pH 7.4)
Which region of Tau protein was fused to yellow fluorescent protein (YFP) for expression in mammalian cells?
Microtubule-binding region (amino acids 243–375)
What was used to seed the Tau monomer in the reactions?
Syringe-sheared Sarkosyl-insoluble material
In which cell type was Tau-YFP expressed by transient transfection?
C17.2 neuronal precursor cells
Study Notes
Cellular Activities of Tau
- Four different constructs were used to characterize the cellular activities of Tau: full-length Tau (441 amino acids) and the microtubule-binding region (amino acids 243-375) fused to yellow fluorescent protein (YFP).
Tau-YFP Expression in Cells
- Tau-YFP was expressed in C17.2 neuronal precursor cells by transient transfection.
- Cells were trypsinized, allowed to recover, and fixed with 4% paraformaldehyde for staining.
- Tau-YFP expression did not show spontaneous aggregation in cells.
Truncated Tau Forms Inclusions
- The microtubule-binding region (MTBR) of Tau formed inclusions in cells.
Co-culture Experiments
- MTBR-YFP and mCherry-expressing cells were co-cultured for 24 hours.
- Co-cultured cells showed a significant increase in YFP/mCherry dual-positive cells (1.6% compared to 0.48% in the control).
- Dual-positive cells were collected and imaged using fluorescence microscopy, confirming the presence of MTBR-YFP inclusions inside cells expressing mCherry.
Inducible Transfer of Tau Aggregates
- Cells expressing full-length Tau-YFP and mCherry were co-cultured and initiated misfolding of Tau-YFP by exposing cells to MTBR Tau aggregates for 48 hours.
- Treatment with aggregated species caused Tau-YFP aggregation and transfer of these species to 1% of the mCherry cells.
Sarkosyl Extraction
- 1% Sarkosyl-insoluble material was purified from cell lysates.
- Sarkosyl-insoluble pellets were resuspended in 10 mM HEPES and 100 mM NaCl (pH 7.4).
Tau Monomer Seeding
- Sarkosyl-insoluble material from cells was used to seed the Tau monomer.
- Reactions contained 4 µM MTBR Tau monomer, 5 mM dithiothreitol, 100 mM NaCl, 10 mM HEPES, and 10 µl of syringe-sheared Sarkosyl-insoluble material.
Test your knowledge on the transfer of intracellular Tau aggregates between co-cultured cells. Understand the process of collecting dual positive cells via FACS, fixing, and mounting them for analysis. Explore the visualization of MTBR-YFP inclusions and mCherry-expressing cells.
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