We performed the analysis of polysome clusters in human cells over 26 images of 16 different cells. Image analysis was performed using the Fiji software. We applied the Bersen auto local threshold plugin to the images to isolate the fluorescent particles. The parameter was chosen to set as positive the pixels that have a gray level higher than the half of the local maximum. For each closed region of positive pixels, the plugin allows the measurement of the shape descriptors and the ellipse fit of the particles.
Finally, we plotted the distribution of the major we called length and minor we called width axis of the ellipse, the area, the aspect ratio, and the solidity of the analyzed particles.
Preparations of polysomes were plunge-frozen on mesh lacey carbon grids in liquid ethane. EM of polysomes for single-particle analysis was performed using an FEI F30 cryo-microscope, capturing images at keV at 36, magnification on SO film. This 3D map was then used to interpret electron tomograms of the same sample and for visualization of the relative orientations of ribosomes within polysomal assemblies.
Sections through the map are shown in Fig. Electron tomograms of polysomes dosed with nm gold markers to act as fiducials in alignment were collected using a Polara cryo-microscope FEI operating at keV and fitted with a charge-coupled device camera for data capture Ultrascan SP; Gatan. This method allows identification of different classes according to their rotational symmetry. The most diverse groups of rotational spectra tend to be pushed toward the corners of the KerDenSOM output map.
The images belonging to each corner for each sample considered LMW, MMW, and HMW polysomes were used to produce a gallery of polysomes with defined rotational characteristics. The diversity between the groups identified by the KerDenSOM output maps was further evaluated by MMD, a powerful nonparametric two-sample test determining whether two samples are drawn from different distributions Gretton et al.
For each sample, the 10 pairwise comparisons with the highest statistic all statistically significant were graphically arranged as heat maps. S1 shows controls of AFM imaging: ribosome assemblies disappear after EDTA and puromycin treatments and display the expected height when measured in liquid. S4 shows the cryo-EM and cryo-ET ribosome and a gallery of polysomes.
Video 1 shows the gallery of a 3D collection of composite polysomes with naked RNA as 3D objects where polysomes show subclusters of ribosomes. We thank W. Filipowicz and R. Mendez for fruitful discussions. Native human polysomes observed by AFM preserve their in vivo organization and reveal tight ribosome interactions. The absorbance peaks corresponding to 40S and ribonucleoparticles are not shown for clarity.
The 80S fraction data look to be grouped in a single cluster, which is also present in the other three fractions, alongside a second cluster that displays an increase of width and length along the gradient. Actin is used as a control for nonribosomal fractions and does not cosediment with ribosomes, ribosomal subunits, and polysomes. The image shown is from a single representative experiment out of three repeats. For the further analysis, we used nine images.
The amount of noise was calculated measuring the mean standard deviation of counts in the background of the STED images. The zoom has been chosen to match the size of the AFM image. The blue lines show the perimeter of the objects identified. The two graphs are normalized by the whole number of counted objects, respectively, and show a good matching.
For each sample under consideration, manual object selection was used to generate galleries of images. Bars, nm. The largest differences thicker stripes are found between classes and and between and corresponding to the most frequent classes in b. Classes and show a slightly lower difference.
Each set of images corresponding to the three most populated classes i. Three recurrent classes of polysomes display specific conformations. Different ribosome—ribosome orientations in HMW polysomes.
Large polysomes are formed by ribosome clusters connected by naked RNA. To appreciate both ribosome and RNA height levels, the image is shown using different Z-ranges for the color scale 0—0. Cross-section profiles solid white line of RNA connecting adjacent ribosome clusters are shown with corresponding height profile compatible with free RNA. Three classes appeared to be the most represented and the MMD was performed. Because in this case we are looking for similarities rather than dissimilarities, we plotted the discrepancy values as negative, so the higher the value the higher the similarity.
Frequency changes of the three cliques after translation inhibition by rapamycin. Cells were fixed and incubated with fluorescent alkyne to label the AHA incorporated into nascent proteins. The kinetics of protein synthesis inhibition open squares and the corresponding decrease in polysome content closed circles are shown. The corresponding distributions of the numbers of ribosomes per transcript are shown. The classification has been performed on and objects purified from MCF7 polysomes before and after rapamycin treatment.
Protein inhibition is relative to the control no rapamycin and was determined by AHA incorporation. Frequency changes of the three cliques are reversible. After 12 h of serum addition, polysomes obtained from serum-starved cells and after FBS readdition were adsorbed on the mica and imaged by AFM. The data shown are from a single representative experiment out of three repeats. Galleries of unclassified HMW polysomes are shown.
Sign In or Create an Account. Advanced Search. User Tools. Sign In. Skip Nav Destination Article Navigation. Article February 23 Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes Gabriella Viero , Gabriella Viero. Correspondence to Gabriella Viero: gabriella.
This Site. Google Scholar. Lorenzo Lunelli , Lorenzo Lunelli. Andrea Passerini , Andrea Passerini. Paolo Bianchini , Paolo Bianchini. Robert J. Gilbert , Robert J. Toma Tebaldi , Toma Tebaldi. Alberto Diaspro , Alberto Diaspro. Cecilia Pederzolli , Cecilia Pederzolli.
Alessandro Quattrone Alessandro Quattrone. Author and Article Information. Gabriella Viero. Lorenzo Lunelli. Andrea Passerini. Paolo Bianchini. Toma Tebaldi. Alberto Diaspro. Cecilia Pederzolli. Alessandro Quattrone. Abbreviations used in this paper:. Received: June 13 Accepted: January 23 Online Issn: J Cell Biol 5 : — Article history Received:. Cite Icon Cite. The authors declare no competing financial interests. Search ADS. Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly A -tail: a cryo electron tomography study.
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