Abstract
Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20–40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50–70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.
Highlights
Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton
We develop single-chain antibody fragments against tubulin and achieve super-resolution imaging of microtubules with a decreased apparent diameter, allowing us to optically resolve bundled microtubules
We found that individual microtubules were densely labelled with the most common diameter varying from 39.3±0.8 nm (VHH#2, N 1⁄4 10 data sets with in total n 1⁄4 1,365 profiles) to 54.0±1.2 and 61.7±0.8 nm
Summary
Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. Single-molecule localization microscopy (SMLM) provides selectivity at an increased resolution, but the extremely small spacing between neuronal microtubules (20–70 nm)[2] poses novel challenges, because existing labelling strategies typically increase the apparent microtubule diameter by 20–40 nm and will thereby blend neighbouring microtubules into one structure[3]. It is widely assumed that despite all progress in super-resolution microscopy, electron microscopy is still the only technique that allows insight into complex microtubule structures[4] We use both computer simulations and experimental approaches to explore how labelling strategy affects SMLM imaging of microtubules. We develop single-chain antibody fragments (nanobodies) against tubulin and achieve super-resolution imaging of microtubules with a decreased apparent diameter, allowing us to optically resolve bundled microtubules
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