Axonal endoplasmic reticulum tubules control local translation via P180/RRBP1-mediated ribosome interactions.

Local mRNA translation in axons is critical for the spatiotemporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons; however, how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation in developing rat hippocampal cultured neurons. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with axonal ER tubules in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation and binds to mRNAs enriched for axonal membrane proteins. Importantly, the impairment of axonal ER-ribosome interactions causes defects in axon morphology. Our results establish a role for the axonal ER in dynamically localizing mRNA translation, which is important for proper neuron development.

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains.

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.

SOLEIL: single-objective lens inclined light sheet localization microscopy.

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 µm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination.

Self-assembly of pericentriolar material in interphase cells lacking centrioles.

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein-mediated PCM transport, but the self-assembly properties of PCM components in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based transport and self-clustering of PCM proteins are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase self-assembly of PCM components depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152, or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor- and microtubule-based rearrangement.

ER - lysosome contacts at a pre-axonal region regulate axonal lysosome availability.

Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the endoplasmic reticulum (ER) and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule - lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule - lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function.

Quantitative mapping of dense microtubule arrays in mammalian neurons.

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport.

Cells in the body need to control the position of the molecules and other components inside them. To do this, they use a system of proteins that work a bit like a road network. The 'roads' are tubular structures known as microtubules, while 'vehicles' are transporters, called motor proteins, that 'walk' along the microtubules. Microtubule networks are important in all cells, but especially in neurons, which can grow very large. These cells have tree-like branches called dendrites that receive messages from other neurons. Dendrites contain different types of microtubules with many chemical modifications. These modifications consist of specific molecules or 'groups' becoming attached to or removed from the microtubules to change their properties ­ for example, microtubules can be 'acetylated' or 'detyrosinated'. Motor proteins prefer different kinds of microtubules, and so understanding transport inside cells involves creating a precise roadmap showing how many of each type of microtubule exist and where they go. Using different super-resolution microscopy techniques, Katrukha et al. created maps of the microtubules in rat neurons. These show that acetylated microtubules form a core in the centre of the dendrites, while tyrosinated microtubules (which did not undergo detyrosination) line the cell membrane of the dendrites. Katrukha et al. then used the maps to determine that acetylated microtubules account for 65 to 70% of all microtubules, while tyrosinated microtubules make up 20 to 30%. This means that most microtubules fall into these two categories. The work by Katrukha et al. provides one of the first quantitative estimates of the relative amount of acetylated and tyrosinated microtubules, starting to shed light on how cells control their transport network. This could ultimately allow researchers to explore how transport changes in health and disease.

Mapping the neuronal cytoskeleton using expansion microscopy.

Expansion microscopy (ExM) is a recently introduced technique that enables high-resolution imaging with conventional microscopes by using physical expansion of samples. While this technique does not require a complicated microscope setup (like in STED or STORM microscopy), sample preparation and handling require additional attention. Here we describe a workflow for imaging of the neuronal microtubule network with minimal artifacts and sample perturbations. We demonstrate that the use of custom-printed mounting chambers simplifies sample handling and facilitates stable imaging of the sample. In addition, refractive index matching between the sample and the objective greatly improves signal retention deeper in thick samples. To accurately determine the precise expansion factor and determine sample distortion, we describe how samples can be compared using STED and ExM. Together, these procedures enabled us to better resolve different microtubule subsets in neuronal soma and dendrites.