SummaryMicrotubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly oriented minus-end out, while dynamic and tyrosinated microtubules are oriented oppositely. In addition, microtubules with similar orientations and modifications form bundles that bias transport. Importantly, because the plus-end-directed Kinesin-1 selectively interacts with acetylated microtubules, this organization guides this motor out of dendrites and into axons. In contrast, Kinesin-3 prefers tyrosinated microtubules and can enter both axons and dendrites. This separation of distinct microtubule subsets into oppositely oriented bundles constitutes a key architectural principle of the neuronal microtubule cytoskeleton that enables polarized sorting by different motor proteins.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

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.

Single-molecule localization microscopy (SMLM) enables fluorescent microscopy with nanometric resolution. While localizing molecules close to the coverslip is relatively straightforward using high numerical aperture (NA) oil immersion (OI) objectives, optical aberrations impede SMLM deeper in watery samples. Adaptive optics (AO) with a deformable mirror (DM) can be used to correct such aberrations and to induce precise levels of astigmatism to encode the z-position of molecules. Alternatively, the use of water immersion (WI) objectives might be sufficient to limit the most dominant aberrations. Here we compare SMLM at various depths using either WI or OI with or without AO. In addition, we compare the performance of a cylindrical lens and a DM for astigmatism-based z-encoding. We find that OI combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep (>10 µm) 3D localization.

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantummechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores. fluorescent proteins | mCherry | localization microscopy | β-mercaptoethanol | photoactivation