Peering at Brain Polysomes with Atomic Force Microscopy

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

The synthesis of protein is the most energy consuming process in cells^1,2. Hence, it is not surprising that protein abundances are mainly controlled at the translational rather than transcriptional level^3,4,5. 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 converge^6-13. Despite hundreds of studies on ribosome structure^{14- 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 nucleosome^17,18.

Since the discovery of the ribosome^14-16, its structure has been extensively characterized in prokaryotes^19,20, 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 systems^23-26, human cellular lysates²⁷ or in cells²⁸. These techniques offered more refined information about the ribosome-ribosome organization in polysomes^{23, 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 eukaryotes^29-33 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 polysome^{27, 35}.

Protokół

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

1. Gathering brain tissues (15 min)
   1. Euthanize wild-type mouse strain C57BL/6 with CO₂ asphyxiation for 5 min³⁶. Carefully dissect the brain out from the skull³⁶, place the tissue into a 1.5 ml tube and immediately place it in liquid nitrogen. Store at -80 °C until use.
2. Preparation of lysate (30 min)
   1. Pulverize the whole brain tissue using a mortar and pestle under liquid nitrogen.
   2. Transfer about 25 mg powder to a cold microcentrifuge tube and immediately (to avoid the thawing of the pulverized tissue) add 0.8 ml of Lysis Buffer (see Table 1) and disrupt the cell by pipetting up and down 25 times quickly.
   3. Centrifuge the tube at 12,000 x g for 1 min at 4 °C to pellet cellular debris.
   4. Transfer the supernatant to a new microcentrifuge tube and keep the tube on ice for 15 min.
   5. Centrifuge the tube at 12,000 x g for 5 min at 4 °C to pellet nuclei and mitochondria.
   6. Transfer the supernatant to a new microcentrifuge tube.
   7. Store the supernatant at –80 °C for a maximum of 6 months or use it immediately.
3. Sucrose gradient preparation and centrifugation (2 hr and 30 min)
   1. Wash ultracentrifuge tubes extensively with RNase-free water (diethylpyrocarbonate treated water (DEPC-water) or commercial) and 3% H₂O₂/DEPC H₂O solution.
   2. Put the tubes on ice and add 5.5 ml of cold 50% sucrose solution at the bottom of each tube (see Table 1 for sucrose solutions). Carefully add the 15% sucrose solution drop by drop, staying close to the interphase in order to preserve a sharp interphase, until the tube is completely filled. When the tube is completely filled, close it with a rubber stopper to avoid air bubble formation.
   3. In a cold room gently lay down the tubes horizontally and keep it in this position for 2 hr. After this time, slowly straighten the tubes back into the vertical position and put them on ice. The gradients are now ready to be used. Alternatively, prepare the 15-50% sucrose gradient using a conventional gradient former.
   4. In a cold room remove carefully 1.0 ml from the top of the gradient and overlay the sucrose drop by drop with the cytosolic lysate (i.e., the supernatant obtained in step 1.2).
   5. Carefully lower the tubes into the buckets of swinging bucket rotor. Centrifuge the gradients for 100 min at 180,000 x g at 4 °C using an ultracentrifuge.   
   6. After the centrifugation, leave the tubes in their buckets for 20 min at 4 °C to let the gradients stabilize.
4. Sucrose gradient fractionation (2 hr)
   1. Carefully remove one ultracentrifuge tube from the ultracentrifuge rotor and mount it on the collector device of a Density Gradient Fractionation System. Collect 1 ml fractions monitoring the absorbance at 260 nm with a UV/VIS detector (See Figure 1 upper panel). Keep the collected fractions on ice.
   2. Prepare aliquots of 30-40 μl of the fractions of interest, keep them on ice before storing them at -80 °C till use. Do not use aliquots or sucrose fractions that underwent more than two freezing-thawing cycles (see Figure 1 lower panel).

2. Sample Preparation for Atomic Force Microscopy (3 hr)

1. Using tape, peel off the mica sheets.
2. Wash the mica sheets 3-4 times with DEPC-water and place it into a small Petri dish. Then dry the surface using air.
3. Cover the mica with 200 μl of 1 mM NiSO₄ and incubate for 3 min at RT.
4. Remove the nickel solution and then dry the surface using air. Carry out all future steps at 4 °C by placing the Petri dish with the mica on ice.
5. Thaw an aliquot obtained in 1.4.2 on ice and gently add all of the sample drop by drop on the mica. Using a 100-200 μl tip, spread the sample on the entire surface of the mica. Incubate the sample on ice for 3 min.
6. Cover the mica sheet drop by drop with 200 μl cold Buffer-AFM (see Table 1) and incubate for 1 hr on ice.
7. Imaging in liquid
   1. Remove carefully the Buffer-AFM and wash the mica sheets 3-4 times with 200 μl cold Buffer-AFM to remove excess sucrose. Then wash the mica sheets 3 times with cold Washing Solution (see Table 1), leaving the mica surface covered by some microliters of solution.
   2. Go to point 3 (Image acquisition).
8. Imaging in air
   1. Carefully remove the Buffer-AFM and wash the mica 3-4 times with 200 μl cold Buffer-AFM to remove the excess sucrose. Then, wash the mica 3 times with cold Washing Solution (see Table 1) and drain the excess water using paper.
   2. Leave the sample to dry under the chemical hood with the top of the Petri dish partially open. After 2 hr, close the Petri dish and store at RT. Measure the sample after 2-3 hr as they are stable for years.

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.

1. Attach the mica to the sample holder using double-sided tape.
2. Insert the sample holder in the AFM stage following manufacturer's directions. When imaging in liquid, if possible, try to maintain the sample at a temperature lower than 25 °C to increase polysome stability in time.
3. Select a cantilever suitable for AC imaging and mount it on the tip holder following manufacturer's directions. Here, use cantilevers with force constant between 2-20 N/m for air imaging and around 0.1 N/m for liquid imaging.
4. Adjust the laser spot on the cantilever and zero the quadrant detector signals.
5. Select an opportune driving frequency and drive the cantilever with an amplitude of 10-20 nm.
6. Approach the sample until the tip engages the surface.
7. Select a scan area of 2x2 μm, acquire at least 512x512 pixel images (pixel width < 4 nm), select a live background subtraction mode and a Z scale of 20-25 nm.
8. Inspect the image looking for the presence of round objects characterized by the height between 10 and 15 nm when acquiring in air and 25 and 30 nm when in liquid and the width in the range 25-30 nm. Adjust the setpoint and feedback parameters until sharp objects are visualized. The background should appear relatively flat in good samples, with some 2-4 nm height objects (see Figure 2A and B).
9. If the image looks good (as indicated at point 3.8), acquire several (at least ten) 2x2 micron scans at different sample areas.
10. (optional). If necessary, acquire high resolution images of selected polysomes (see Figure 2C).
11. Apply software corrections (plane subtraction and line-by-line correction algorithms) to correct the AFM images removing arbitrary tilt and drifting effects.

4. Data Analysis (30 min per image)

1. Export images in ImageJ (optional: apply a scale factor) preferentially using a lossless compression format, e.g., the TIFF format (see Figure 3A).
2. Use the ImageJ macro toolset RiboPick.ijm (to be copied in the ImageJ macro/toolset subdirectory) to count ribosomes in polysomes (see Figure 3B) and compute statistical properties of the sample (see Figure 3C).
   1. Start loading an image using the <Read image> tool that initializes the program.
   2. Pick the ribosome centers with the standard ImageJ <Point selection> tool (shift + left click allows the multi-selection of ribosomes). Mark the selected ribosomes with the <Mark ribosomes> tool. Ribosome coordinates will appear in a custom text window.
   3. Add more ribosomes to the same polysome repeating the procedure indicated at the point 4.2.2.
   4. Remove wrongly marked ribosomes using the <Undo last pick> tool (starts removing from the last added ribosome to the first one — in the current polysome only).
   5. When the polysome is completed, use the <New polysome> tool. As the polysome number is added as an overlay to the image, the text window is updated.
   6. Use the <Save results and close> to write the ribosome coordinates file (the default units of the image are used) and a PNG image that summarizes the picked ribosomes and polysomes. The original image is closed without being saved.

Wyniki

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 ...

Dyskusje

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. ...

Materiały

Name	Company	Catalog Number	Comments
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