While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.
The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.
The synthesis of protein is the most energy consuming process in cells1,2. Hence, it is not surprising that protein abundances are mainly controlled at the translational rather than transcriptional level3,4,5. The polysome is the fundamental macromolecular component that converts the mRNA information into functional proteinaceous readouts. Polysomes are thus far recognized as macromolecular complexes where several translational controls converge6-13. Despite hundreds of studies on ribosome structure14- 16, the detailed molecular insights into the dynamics of translation and the topology of polysomes has encountered a limited interest. As a consequence, the organization of the native polysomal ribonucleoprotein complex and its potential effect on translation are still rather obscure issues. Polysomes may hide still unknown ordered and functional organizations, potentially mirroring what nucleosomes and epigenetic controls have represented for the transcription field. Indeed, the investigation of this intriguing hypothesis requires additional studies and new technical approaches. In this line, structural techniques and atomic force microscopy may fruitfully collaborate to unravel new mechanisms for controlling gene expression, similarly to what was achieved for the nucleosome17,18.
Since the discovery of the ribosome14-16, its structure has been extensively characterized in prokaryotes19,20, yeast21 and more recently in human22, providing the molecular description of the mechanisms at the base of protein synthesis. Polysomes were initially recognized on the membrane of the endoplasmic reticulum, forming typical 2D geometrical organizations14. As mentioned before, polysome assembly has not been the object of constant interest as the ribosome structure. In the past, polysomes have been studied essentially by transmission EM-based techniques. Only recently, cryo-EM techniques enabled the 3D reconstruction of purified polysomes from in vitro translation systems23-26, human cellular lysates27 or in cells28. These techniques offered more refined information about the ribosome-ribosome organization in polysomes23, 26-28 and a preliminary molecular description of the contact surfaces of adjacent ribosomes in wheat germ polysomes24. Thus, cryo-EM tomography allows the disclosure of ribosome-ribosome interactions with molecular detail, but it is burdened by extensive post-processing and reconstruction analyses that require heavy computational resources for data handling. Moreover, to obtain the molecular detail of ribosome-ribosome interactions, a highly resolved map of the ribosome is required, and this kind of reference ribosome is available only for a few species. Importantly, cryo-EM tomography is not able to detect free RNA. Therefore, new techniques are required to fully understand the organization of the translation machinery.
Beside cryo-EM, AFM has been also employed as a useful tool for direct imaging of polysomes in lower eukaryotes29-33 and humans27. Compared to EM, AFM requires no sample fixation or labeling. In addition, measurements can be carried out in near physiological conditions and with the unique possibility of clearly identifying both ribosomes and naked RNA strands27. Imaging of single polysomes can be performed relatively quickly, obtaining thousands of images at nano-resolution with little post-processing effort compared to the extensive and heavy post-processing and reconstruction analyses required by cryo-EM microscopy. Consequently, AFM data handling and analysis does not need expensive workstations and high computing power. As such, this technique collects information on polysomal shapes, morphological characteristics (such as height, length and width), ribosome densities, the presence of free RNA and the number of ribosomes per polysome27 with a higher throughput than cryo-EM. In such way, AFM represents a powerful and complementary approach to EM techniques to portray polysomes27.
Here we present a complete pipeline from purification to data analysis where AFM is applied to image and analyze mouse brain polysomes. The proposed protocol focuses on purification issues and on the accurate deposition of polysomes on mica substrates that are used for AFM imaging. In addition to conventional particle analysis that can be easily performed with common software used by the AFM community, an ImageJ34 plugin, called RiboPick, is presented for counting the number of ribosomes per polysome27, 35.
The practices used to obtain the mouse tissues were approved by the Body for the Protection of Animals (OPBA) of the University of Trento (Italy), protocol no. 04-2015, as per art.31 Legislative Decree no. 26/2014. All mice were maintained at the Model Organism Facility of the Centre for Integrative Biology (CIBIO), University of Trento, Italy.
Caution: To avoid any RNA degradation of the samples, prepare all buffers using DEPC-treated water for minimizing RNase contamination.
1. Preparation of Polysomes from Whole Brains
2. Sample Preparation for Atomic Force Microscopy (3 hr)
3. Image Acquisition (15 min per image after thermal stabilization)
Note: Polysomes immobilized on mica can be imaged in air or in liquid, using AC mode.
4. Data Analysis (30 min per image)
Sucrose gradient polysomal profiling of whole mouse brain
It is possible to purify polysomes from a cellular lysate by polysomal profiling, which separates macromolecules in accordance to their weight and size. With polysomal profiling, cytoplasmic lysates obtained from cultured cells or tissues, such as in this example, are loaded onto a linear sucrose gradient and processed by ultracentrifugation to separate free RNA from 40S and 60S subunits, 80S ribosomes and polysomes according to their sedimentation coefficients (Figure 1 upper panel). The absorbance of the sample at 254 nm and the fraction collection enable the isolation of the sucrose fraction enriched in ribosomes or polysomes with different number of ribosomes per transcript. The fractions can be collected, aliquoted for AFM imaging and stored at -80 °C (Figure 1 lower panel).
Native polysomes from mouse brain observed by AFM in air reveal tight ribosome interactions
Image acquisition is performed using AC mode (also known as tapping mode). This modality is especially suitable for imaging soft samples (such as the polysomes) because the tip-sample interaction and the corresponding shear forces are greatly reduced. In this way both the tip and the sample are preserved. AFM imaging allows the acquisition of data at high resolution, with exact values that depend on various factors, such as the measuring conditions, the kind of the tip, and the properties of the sample. In the conditions described in this paper a lateral resolution of about 4-6 nm and a vertical resolution of 0.1-0.2 nm are attainable. After the deposition of sucrose aliquots on mica, AFM provides descriptions of single polysomes that appear as clusters of tightly packed ribosomes (Figure 2A, C and C). By cross section analysis (Figure 2D), the height of ribosomal peaks is around 14 nm in accordance to what was previously observed for human ribosomes in polysomes after air drying27. The center to center distance of ribosomes identified by the line profile is 23 nm, which is in agreement with the dimension of ribosomes in solution37, 38.
The number of ribosomes per polysome from a single fraction shows a mono-dispersed distribution
Figure 3A reports a typical AFM image obtained by acquiring a sample absorbed on mica from a fraction of the sucrose gradient. The inset shows a digital zoom of a single polysome, with a magnification factor suitable for the identification of ribosomes in the polysome. Panel B shows the same image after the ribosome identification with the RiboPick macro. Red circles (see the inset) mark the ribosomes, and a progressive number (cyan) is used to help the association of the ribosome coordinates with a specific polysome. The frequency distribution of the number of ribosomes per polysome was analyzed for fraction #10 (See Figure1, lower panel, corresponding to Medium Molecular Weight polysomes (MMW)), which clearly shows a single peak (Panel C, grey bars). The experimental distribution was fitted with a Gaussian curve (black line) that was centered at 5.8 ribosomes/polysome, with a standard deviation of 1.3 ribosomes/polysome.
Figure 1. Representative absorbance profile for sucrose gradient sedimentation of whole mouse brain. (upper panel) A polysomal cytoplasmic lysate was obtained from P27 mouse brain and loaded onto a linear sucrose gradient (15-50% sucrose [w/v]). It is possible to observe clear peaks corresponding to absorbance at 254 nm of RiboNucleoParticles (RNP), ribosomal subunits (40S and 60S), ribosomes (80S) and polysomes. To avoid repeated freeze and thaw cycles of samples before AFM deposition on mica, it is convenient to split into aliquots both ribosome fraction and polysomal fractions (lower panel) with different number of ribosomes per transcript (i.e., low-, medium-, and high-molecular-weight polysomes (LMW, MMW, and HMW, respectively)). The aliquots can be stored at -80 °C for as long as two years.
Figure 2. Images of brain polysomes by Tapping-Mode Atomic Force Microscopy. (A) Example of AFM image of MMW brain polysomes after absorption on mica using a linear grey color scale. (B) The same image shown in (A) is depicted using a different Z-ranges for the color scale: 0-0.5 nm gray for the background, 0.5-2 nm, and 2-10 nm yellow for ribosomes. In this case a better visual contrast is obtained for both low and high Z-range objects. (C) Magnification of a typical MMW polysome with cross-section profile (dotted white line). (D) Height profile of two ribosomes defined by cross-section profile in (C) shows ribosome height of 14 nm. This value is compatible with what previously observed in AFM for human ribosomes in polysomes27.
Figure 3. Example of counting the number of ribosomes per polysome in MMW brain polysomes. (A) AFM image that represents the input of the ImageJ macro, RiboPick. (B) After manually picking each ribosomes per polysome, RiboPick marks in the image the position of each ribosome (red circles). (C) The obtained number of ribosomes per polysomes can be plotted as a distribution that can be fitted with a Gaussian curve. The number of polysomes considered in this example is 164, obtained from 9 independent images. The fitting with the Gaussian curve returns the mean value of the population of polysomes in the MMW fraction (#ribosomes/polysome 5.8±1.3, R2= 0.99).
Buffer | Composition | Application |
Lysis Buffer | 10 mM Tris–HCl, pH 7.5 | Preparation of lysate (1.1) |
10 mM NaCl | ||
10 mM MgCl2 | ||
1% Triton-X100 | ||
1% Na-deoxycholate | ||
0.4 U/ml RNase Inhibitor | ||
1 mM DTT | ||
0.2 mg/ml cycloheximide | ||
5 U/ml Dnase I | ||
50% sucrose solution | 50% (w/v) sucrose in | Sucrose gradient preparation (1.2) |
100 mM NaCl | ||
10 mM MgCl2 | ||
10 mM Tris/HCl pH 7.5 | ||
15% sucrose solution | 15% (w/v) sucrose in | Sucrose gradient preparation (1.2) |
100 mM NaCl | ||
10 mM MgCl2 | ||
10 mM Tris/HCl pH 7.5 | ||
Nickel Solution | 1 mM NiSO4 | Sample preparation for AFM (2) |
Buffer-AFM | 100 mM NaCl | Sample preparation for AFM (2) |
10 mM MgCl2 | ||
100 µg/ml cycloheximide | ||
10 mM Hepes | ||
3% (w/v) sucrose, pH = 7.4 | ||
Washing Solution | DEPC-Water | Sample preparation for AFM (2) |
100 µg/ml cycloheximide |
Table 1. Buffers.
As the structure of DNA was proven to be of paramount importance to describe the process of transcription and as the organization of chromatin advanced our understanding of transcriptional control of gene expression, it is essential to analyze the organization and structure of polysomes to improve the truthful comprehension of translation and its regulation.
With the described protocol, an optimal density of polysomes gently immobilized on a flat surface, suitable for AFM imaging is obtained. Using these conditions with conveniently prepared samples, nearly 50 polysomes per hour can be acquired, ready for further analyses and characterization. Moreover, dried samples stored at RT have proven to be suitable for imaging after more than one year.
To successfully obtain polysome images with our protocol, there are some crucial steps: to use tissues that has been properly flash-frozen immediately after dissection and stored at -80 °C to minimize RNA degradation; to use the lysis buffer containing cycloheximide and RNase inhibitors to avoid ribosome dissociation from mRNA; to perform all incubations on ice during the sample deposition on mica; and when preparing the sample for AFM imaging on air, to extensively wash the sample on the mica with buffers and RNase free water in the presence of cycloheximide. The last point is particularly important. If the image appears to contain smooth surfaces at approximately the right height but 100-1,000 nm wide, this is a clear indication of a poorly washed sample (often ribosomes/polysomes can be still observed as faint, embedded objects). A method to resolve this issue is to repeat the washing procedure, but this does not usually mitigate the issue on an already dried sample. In this case it is worth starting again with a new aliquot.
Given the resolution of AFM, this method for studying polysomes cannot resolve the structural details of ribosomal interfaces in polysomes. In fact, AFM is a complementary and simpler approach to cryo-EM, representing an effective and robust technique, to acquire and analyze thousands of polysomes, and better understand the overall ribosome organization in polysomes. Importantly, this protocol has the unique advantage of identifying filaments compatible with RNA27. This very same procedure can be used for any polysomal sample obtained from any biological tissue or cell line. More importantly it can also be successfully employed to obtain transcript-specific information using in vitro translation systems, as recently demonstrated by Lauria and co-workers35. This methodology will pave the way for several experiments, to gain understanding of the kinetics of polysome formation or changes in polysome organization under different cellular or tissue conditions.
The authors have nothing to disclose.
This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.
Cycloheximide | Sigma | 01810 | Prepararation of lysate |
DNAseI | Thermo Scientific | 89836 | Prepararation of lysate |
RiboLock RNAse Inhibitor | Life technologies | EO-0381 | Prepararation of lysate |
DEPC | Sigma | 40718 | Prepararation of lysate |
Triton X100 | Sigma | T8532 | Prepararation of lysate |
DTT | Sigma | 43815 | Prepararation of lysate |
Sodium Deoxycholate | Sigma | D6750 | Prepararation of lysate |
Microcentrifuge | Eppendorf | 5417R | Prepararation of lysate |
Sucrose | Sigma | S5016 | Sucrose gradient preparation |
Ultracentrifuge | Beckman Coulter | Optima LE-80K | Sucrose gradient centrifugation |
Ultracentrifuge Rotor | Beckman Coulter | SW 41 Ti | Sucrose gradient centrifugation |
Polyallomer tube | Beckman Coulter | 331372 | Sucrose gradient centrifugation |
Density Gradient Fractionation System | Teledyne Isco | 67-9000-176 | Sucrose gradient fractionation |
AFM | Asylum Research | Cypher | Polysome visualization |