Related Experiment Videos
Mutated tau binds less avidly to microtubules than wildtype tau in living cells
E W Nagiec1, K E Sampson, I Abraham
1Cell & Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan 49007, USA. eenagiec@am.pnu.com
Abstract:
Some forms of genetically inherited dementia, including frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused by mutations in tau. We have examined several mutations in the microtubule-binding portion of tau for their effect on microtubule binding, cellular distribution and cytoskeletal structure in mammalian cells. Using constructs coding for mutant (P301L and V337M) and wildtype human tau fused to a green fluorescent protein analog (EGFP) we followed the disposition of tau in live cells after transient transfection using confocal microscopy. Most of the tau protein localized to structures that resembled microtubules or microtubule bundles and co-localized with tubulin. At 3 days post-transfection mutant tau proteins showed a higher abundance of free tau in the cytoplasm than did wildtype tau. Cells expressing the P301L mutation showed proportionally more cytoplasmic localization of tau. Confirming these results, fractionated cells with mutant tau had a higher percentage of tau in the cytoplasmic compartment as compared to the cytoskeletal compartment. Cells with wildtype tau had most tau in the cytoskeletal fraction. Because the mutations (V337M, P301L) are associated with genetic tauopathies, these results suggest that a factor in disease etiology of genetic tauopathies and other dementias with altered tau is a greater abundance of tau in the cytoplasm due to decreased binding to microtubules. This increased cytoplasmic presence may be a significant factor in promoting tau aggregation.
Insights
Mutations in tau protein, linked to inherited dementias, reduce its binding to microtubules. This results in more tau in the cytoplasm, potentially driving disease progression and aggregation.
Area of Science:
- Neuroscience
- Cell Biology
- Genetics
Background:
- Genetically inherited dementias, such as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), can arise from mutations in the tau protein.
- Tau protein plays a crucial role in stabilizing microtubules, essential components of the cell's cytoskeleton.
Purpose of the Study:
- To investigate the impact of specific tau mutations (P301L and V337M) on microtubule binding, cellular localization, and cytoskeletal structure in mammalian cells.
- To understand the cellular mechanisms underlying genetic tauopathies and related dementias.
Main Methods:
- Utilized constructs of wildtype and mutant human tau fused to EGFP for live-cell imaging.
- Employed transient transfection and confocal microscopy to track tau protein disposition in mammalian cells.
- Performed cell fractionation to quantify tau distribution between cytoplasmic and cytoskeletal compartments.
Main Results:
- Mutant tau proteins (P301L, V337M) exhibited reduced binding to microtubules compared to wildtype tau.
- Cells expressing mutant tau showed increased cytoplasmic localization and a higher proportion of free tau in the cytoplasm.
- Wildtype tau predominantly localized to the cytoskeletal fraction, while mutant tau was found in higher percentages within the cytoplasmic fraction.
Conclusions:
- Decreased microtubule binding of mutant tau leads to its increased abundance in the cytoplasm.
- This elevated cytoplasmic tau may be a key factor in the pathogenesis of genetic tauopathies and other tau-related dementias.
- Increased cytoplasmic tau presence could promote tau aggregation, a hallmark of neurodegenerative diseases.