In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 110, No. 4 ( 2013-01-22)
Abstract:
We tackled the challenge of visualizing the fuzzy coat of Tau fibrils by applying high-resolution immunogold-TEM and AFM in the force-volume (FV) mode. For every pixel of the high-resolution AFM image, FV-AFM records a force-distance curve from which the local mechanical deformation and adhesion are extracted. This makes FV-AFM particularly suited to image single biomolecules at high force sensitivity and to map physicochemical properties simultaneously with nanometer resolution (≈1–2 nm). The combination of both high-resolution microscopic techniques allowed us to image and probe the structural properties of the fuzzy coat of single Tau fibrils. Our results reveal that the fuzzy coat formed by short C-terminal and long N-terminal domains of hTau40 fibrils resembles an ≈16-nm-thick, negatively charged, two-layered polyelectrolyte polymer brush (Fig. P1). We further demonstrated that the mechanical and adhesive properties of this brush can change from stiff and repulsive to flexible and adhesive in response to alterations in pH and electrolyte concentration. This responsiveness of the fuzzy coat suggests that changes in the physiological environment, such as those that occur in diseased states, modulate the mechanical stability of Tau fibrils, alter the interactions between Tau fibrils and proteins, and influence the aggregation propensity of Tau fibrils in vitro and in vivo. This information will help to elucidate how the aggregation properties of Tau contribute to neurodegenerative diseases. Previously, we used high-resolution atomic force microscopy (AFM) to observe the structural polymorphism of Tau fibrils in physiological solution ( 5 ). Fibrils of recombinant full-length hTau40 showed an apparent maximum thickness of ≈18.5 nm. Fibril cores assembled from the Tau repeat domain were unexpectedly similar (≈15.9 nm). This suggests that the N- and C-terminal domains in hTau40 fibrils, which represent the majority of the protein mass, contribute only ≈2.6 nm to the fibril thickness. In contrast, if the unstructured termini would collapse onto the fibril core with dense packing of the chains, they should occupy a volume that contributes at least ≈11 nm to the fibril thickness, with an expected overall diameter of ≈30 nm. This raises the question: Which structural and physiochemical properties make the fuzzy coat largely invisible in AFM, TEM, and NMR? The pathological formation of fibrillar aggregates of the microtubule-associated Tau protein is a major hallmark of several neurodegenerative diseases, notably Alzheimer’s disease, Parkinson disease, frontotemporal dementia, and others. In the human brain, six main isoforms of Tau bind and stabilize axonal microtubules. Binding of Tau to microtubules is facilitated by four ≈31-aa-long microtubule-binding pseudorepeats and their flanking regions. The ≈240-aa-long N-terminal domain and the ≈70-aa-long C-terminal domain, which together make up ≈70% of the longest human Tau protein, hTau40, remain unstructured ( 1 ) and project from the microtubule into the surrounding medium. Dissociation of Tau from microtubules is induced by phosphorylation of Tau at multiple sites by various kinases ( 2 ). Once dissociated from microtubules, Tau can assemble into fibrils, which appear in transmission EM (TEM) images as paired helical filaments ( 3 ). Whereas the short β-stranded hexapeptide motifs facilitate the aggregation of Tau repeat domains in the fibril core, the much longer and largely unstructured N-terminal and C-terminal domains are thought to protrude from the fibril core. This model, in which the core of a fibrillar Tau aggregate is surrounded by a so-called “fuzzy coat,” was inferred from early TEM images of antibody-labeled Tau termini that partially projected from the fibril ( 4 ). However, the structural heterogeneity of the Tau termini has thus far limited the characterization of the fuzzy coat by TEM and NMR. Consequently, various properties of the fuzzy coat have not been quantified. Revealing insight into the properties of the fuzzy coat is of importance to understand how Tau fibrils disturb cellular interactions and accumulate in neurofibrillary tangles.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1212100110
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2013
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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