Resolving bundled microtubules using anti-tubulin nanobodies

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.

Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

“…, rat anti-tubulin YL1/2). 36,37 In contrast, the anti-acetylated tubulin primary antibody that we used (mouse anti-acetylated tubulin, clone 6–11B-1) binds to an epitope on the inner surface of microtubules 39,40 which likely leads to a narrower distribution. The narrow FWHM values may also be influenced by the unique structure of Giardia’s adhesive disc, where microtubules are bound on several sides by disc microribbons and other structures 29 that may help to confine immunolabeling to a narrow region.…”

“…36,37 Although microtubules are well-known to have a ~25 nm outer diameter, 38 microtubules labeled with primary and secondary antibodies are substantially broadened when using primary antibodies which bind the outer surface of microtubules ( e.g. , rat anti-tubulin YL1/2).…”

Periodic F-actin structures shape the neck of dendritic spines

“…STochastic Optical Reconstruction Microscopy, STORM, etc.) and Stimulated Emission Depletion (STED) nanoscopy offer a resolution down to 10–50 nm and 35 nm, respectively, and allow imaging of fixed or living samples15161718. In combination with the highly specific protein labelling associated with multi-color fluorescence microscopy, these methods are well suited to study the properties of the dense neuronal cytoskeleton.…”

Super-resolution imaging in glycoscience: New developments and challenges

“…57 Single-chain antibody fragments (nanobodies) against tubulin was engineered to achieve super-resolution imaging of microtubules with a decreased apparent diameter. 58 In parallel, aptamers (short single stranded oligonucleotides ($ 15 kDa, $ 4 nm), as small as nanobodies, can also bene¯t the STED imaging when compared with antibody staining, 59 as seen in Fig. 3.…”