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. 2021 Jul 20;12:4393. doi: 10.1038/s41467-021-24675-8

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© The Author(s) 2021

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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Fig. 3 — a Left: Schematic illustration of the polymerization reaction and subsequent capping of the filament ends. Filament polymerization is induced by addition of DNA oligonucleotides that bridge barrel monomer helical interfaces. Subsequently, capping objects are added to solution which feature single-stranded DNA overhangs complementary to barrel ends. Cap attachment quenches further polymerization. The piston (green object inside the filament) is now sterically trapped. Right: Laser-scanned image of an agarose gel (2%, 21 mM MgCl₂, 90 V, 90 min, ice-water bath) on which the following samples were electrophoresed: L = 1 kb ladder, A = closed barrel, B = capping object, AB = closed barrel-capping object 1 dimers, C = closed barrel-capping object 1 dimers, C = capping object 2, AC = closed barrel-capping object 2 dimers. The total number of conducted gel-electrophoretic analyses of samples prepared following the same protocol as illustrated here (steps 10 and 11 in the transport system sample preparation protocol) was 3 times and showed similar results. b Exemplary negative-staining TEM image of a capped filament. Green arrow: piston. Insets highlight cap monomers. Scale bar: 100 nm. The total number of conducted TEM analyses of samples prepared following the same protocol as illustrated in the transport system sample preparation protocol was 5 times and showed similar results. c Left: Typical field of view negatively stained TEM image of polymerized filaments. Right: Typical field of view TIRF image of polymerized filaments with piston objects trapped inside. The filaments are labeled with Cyanine-5 dyes (10 per barrel monomer), the piston carries 8 Cyanine-3 dyes, image is merged from the two fluorescence channels. Scale bars: 1 μm. The total number of conducted TEM analyses of samples prepared following the same protocol as illustrated in the transport system sample preparation protocol exceeded 10 times and showed similar results. d Exemplary sequence of single frames taken from a TIRF movie reflecting movement of a piston along a filament. Bottom: Standard deviation from the mean image for the entire movie (6000 frames, frame rate = 10/s), illustrating that the piston has traveled across the entire length of this ~3 μm long filament. Scale bar: 1 μm. Source data are provided as a Source Data file.