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. 2017 Nov 27;7:16386. doi: 10.1038/s41598-017-15575-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Characterization of αSyn binding to MTs using colloidal gold particles and Halo-tags. (a,b) αSyn binding to MTs was analyzed by transmission electron microscope (TEM). MTs were mixed with colloidal gold-labeled αSyn (Gold-αSyn) and negatively stained with 2% of uranyl acetate. Gold-αSyn was prepared from N-terminal His-tagged αSyn (His-αSyn). Gold-αSyns appeared as string-like αSyn polymers on MTs. (c) TEM image of MT polymerized with Halo-tagged αSyn (Halo-αSyn). Bamboo joint-like structures (indicated by magenta arrowheads) are visible on the MTs. (d) MT end structure with Halo-αSyn. Joint-like structures similar to those shown in (c) are indicated by magenta arrowheads. Halo-αSyns were also observed in the zone between the outwardly opened tubulin sheet and MT cylinder (blue arrowheads). (e) Gold-αSyn located at the transition zone (blue arrowheads). (f) MT pull-down assay. mNudC co-precipitated with MTs was examined in the absence and presence of αSyn. (g) Dual labeling immunoelectron microscopy (IEM) used to visualize the interaction of MT with mNudC and αSyn. mNudC was labeled with 10 nm colloidal gold (green) via anti-mNudC antibody; and His-αSyn was labeled with 5 nm colloidal gold (red). Co-localization of mNudC and αSyn on a MT is indicated by arrowheads. (h) MT polymerized with Halo-αSyn (magenta) and Gold-mNudC (green). Bamboo joint-like structures (magenta) and colloidal gold (green) indicate co-localization of mNudC with Halo-αSyn on a MT. (i) Cryo-TEM image of MTs polymerized with Halo-αSyn. Joint-like structures on MTs are indicated by magenta arrowheads. MT pfs numbers determined from Moiré patterns are indicated at the top right. (j) Distribution of the pfs numbers of polymerized MTs. The MTs assembled from 40 μM of tubulin (tu) without paclitaxel stabilization mainly formed 13- and 14-pfs MTs (for tu 40 μM, N = 261). The addition of αSyn clearly increased the number of MTs carrying 14-pfs even at 5 μM tubulin (tu 40 μM + αSyn, N = 259; tu 40 μM + Halo-αSyn, N = 135; tu 5 μM + αSyn, N = 111). (k) Cryo-TEM image of MTs polymerized with αSyn and 5 μM of tubulin. MT pfs numbers determined from Moiré patterns are indicated at the top right. Bar: 30 nm. (l) Selective binding of αSyn to MTs. A mixture of axoneme-nucleated MTs (axoneme-MTs) and GMPCPP polymerized MTs (GMPCPP-MTs) was incubated with TMR-Halo-αSyn (red) in a chamber. The white arrows indicate axoneme-MTs; narrow MTs correspond to GMPCPP-MTs. TMR-Halo-αSyn appears to preferentially bind to GMPCPP-MTs, but not to axoneme-MTs. Scale bar: 30 nm in (a–e), (g–i) and (k); and 5 μm in (l).