Biophysics

Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture.

Micro-computed tomography (micro-CT) is a powerful, non-destructive imaging technique that creates high-resolution 3D images of the internal structures of small animal models such as mice and rats. Familiarizing oneself with micro-CT imaging and data analysis can be overwhelming without easy-to-follow, clear instructions. Training on new instruments is often a task exclusive to a select subset of researchers, leaving the majority of potential trainees without a technical grasp of how to navigate the instructions. This protocol on the use of micro-CT aims to bridge that gap by providing a clear, step-by-step guide to acquire and analyze micro-CT images from mice for quantitative data. By exclusively detailing the necessary procedural steps from start to finish and overcoming complex user interfaces during imaging operations and analysis, this protocol will equip new micro-CT users with the ability to measure mouse body composition (bone, body fat, and lean muscle mass) and identify and quantify lung fibrosis. This approach applies to researchers with a basic understanding of medical imaging, animal care, and software analysis.

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples.

Key features

• Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET).

• Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms.

• The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.

Graphical overview

Hepatitis B virus (HBV) infection is a global public health concern. During chronic infection, the HBV small-surface antigen is expressed in large excess as non-infectious spherical subviral particles (SVPs), which possess strong immunogenicity. To date, attempts at understanding the structure of HBV spherical SVP have been restricted to 12–30 Å with contradictory conclusions regarding its architecture. We have used cryo-electron microscopy (cryo-EM) and 3D image reconstruction to solve the HBV spherical SVP to 6.3 Å. Here, we present an extended protocol on combining AlphaFold2 prediction with a moderate-resolution cryo-EM density map to build a reliable 3D model. This protocol utilizes multiple software packages that are routinely used in the cryo-EM community. The workflow includes 3D model prediction, model evaluation, rigid-body fitting, flexible fitting, real-space refinement, model validation, and model adjustment. Finally, the described protocol can also be applied to high-resolution cryo-EM datasets (2–4 Å).