Peering at Brain Polysomes with Atomic Force Microscopy

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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....

Results

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 .......

Discussion

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. .......

Materials

List of materials used in this article

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