Ribosome-Associated Vesicles promote activity-dependent local translation

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics , identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation

3D Architecture of the <i>Trypanosoma brucei</i> Flagella Connector, a Mobile Transmembrane Junction

<div><p>Background</p><p>Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist <i>Trypanosoma brucei</i>, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.</p><p>Methodology/Principal Findings</p><p>We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.</p><p>Conclusions/Significance</p><p>The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.</p></div

The 3D architecture of the FC region as showed by electron tomography.

<p><b>A)</b> Tomographic slice of a chemically fixed sample showing the tri-laminar structure, its partition into plates and filamentous connectors to the two axonemes (arrows). <b>B)</b> 3D model of the two flagella in the FC region. The FC tri-laminar layer is shown in green (old flagellum layer), gold (interstitial zone layer), turquoise (new flagellum layer). The flagellar membranes are shown in pink, the central pair in green and the doublet microtubules are colour-coded according to the gradient shown below the models. <b>C)</b> 3D models of the axoneme near the flagella connector. The filamentous network is shown in gold and black. <b>D)</b> A 3D model of the tri-laminar structure first illustrated as a whole, and then separated into the individual layers. Numbers by the dotted lines show the number of plates that would be seen in a FC cross-section at this point. <b>E)</b> The orientation of the plates in the old and new flagellum FC layers are indicated by black lines. These lines appear parallel to the MTs of the underlying axoneme. <b>F</b>) A tomographic slice of a high pressure frozen flagella connector. The old flagellum and interstitial zone layers appear as continuously electron dense, instead of being partitioned into plates. In the new flagellum no layer is discernable, instead there is an electron dense cap. G) 3D model of the FC region (same color codes as above). Note the close proximity of the axoneme to the smooth flagellar membranes. H) In high pressure frozen samples no filamentous network is observable between the tri-laminar structure and the axonemes. I) Illustration of the three layers of the FC in a high pressure frozen cell.</p

The flagella connector (FC) is a motile cellular junction spanning the membranes of both the old and new flagellum.

<p>A) Scanning EM micrograph of a procyclic T. brucei in which the new flagellum has exited the flagellar pocket and is attached to the old flagellum at the FC (see higher magnification image of this area). B) A cartoon adapted from [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004312#pntd.0004312.ref017" target="_blank">17</a>] showing the known structure of the FC, as if seen in cross-section. C) A chemically fixed FC in cross section, the FC appears as electron dense layers in the new flagellum (NF) and old flagellum (OF), as well as in an interstitial zone. D) In high pressure frozen cells the FC is less apparent, the new flagellum is more electron dense and the flagella are round in profile. E) Cryo-EM of frozen hydrated sections of a FC shows a repetitive structure in the interstitial zone. F-F’) A zoomed in image of the structure in E. In F’, breaks in the repetitive interstitial densities are highlighted (blue transparent boxes).</p

Structural maturation of the FC in high pressure frozen cells.

<p><b>A)</b> A 3 nm thick tomographic slice of a forming FC on the side of an old flagellum. No physical connections to the 0.65 μm long new flagellum are observable at this stage. <b>B)</b> A 3 nm tomographic slice of a mature FC (7.7 μm long new flagellum). Note the thick electron dense layer inside the old flagellum. <b>C)</b> The interstitial zone thickness, as measured at its thinnest point, correlated with the new flagellar length. Note its thinning as the FC matures. <b>D-F)</b> The old flagellum FC layer thickness, the flagella connector length and depth seems variable but with no obvious correlation to cell cycle progression.</p

Dimensions of the FC in different sample preparation methods.

<p>Dimensions of the FC in different sample preparation methods.</p

Cryo-electron tomography of a FC reveals linear, periodical protein arrangements in the interstitial zone.

<p><b>A)</b> An overview of the cryo-section shows the old flagellum, new flagellum tip and the cell body sectioned transversally to the cells longitudinal axis. The electron dense particles close to the FC are of unknown nature and discussed further in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004312#pntd.0004312.s002" target="_blank">S2 Fig</a>. <b>B)</b> The objects of interest were traced on the tomographic slices: microtubules (green), the old flagellum (blue), new flagellum (gold), cell body (beige) and the densities found in the FC region (red) <b>C)</b> A 3D model of the tomogram reconstruction. Note the small diameter and cupped morphology of the new flagellum in comparison to the old flagellum, which indicates that this is the distal-most tip of the new flagellum. <b>D-F)</b> 15 nm thick slices from the tomogram reconstruction, note the increasing diameter of the new flagellum in E-F, as well as its decreasing distance to the old flagellum. This shows that the new flagellum distal tip is further from the old flagellum than the more proximal parts. In <b>D)</b> 3 layers of stacked electron dense periodical structures are found between the NF and OF. In <b>E)</b> this is reduced to two layers and in <b>F)</b> only one layer is seen. <b>G)</b> The periodical structures seen in E-F are parallel lines 7 nm apart when tilted 60° in x. These lines have periodically darker areas every 11 nm as indicated by the black arrowheads.</p

The FC displaces the old flagellum axoneme and its position is fixed in relation to both axonemes.

<p><b>A)</b> 3D reconstructions of the FC region show it in close proximity to microtubule doublets 3–5 in the new flagellum and microtubule doublets 1, 7–9 in the old flagellum both in high pressure frozen and chemically fixed samples. <b>B)</b> A cartoon illustrating the FC and the axoneme orientations in cross-section. <b>C)</b> An example tomographic slice of a 30 nm thick flagellum cross-section, oriented with the microtubule quartet to the left, shows the axoneme located in the top-left corner of the flagellar space. <b>D)</b> The line drawing shows a few examples of axoneme positioning within the old flagellum (black ellipsoids), and at the flagella connector (red ellipsoids). <b>E)</b> The distances between the FC and the nearest doublet microtubule are longer in the old flagellum.</p