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Advanced Freeze-Replica Methods for Virus Cell Biology

¹Institute of Materials and Systems for Sustainability, Nagoya University, Nagoya, Japan. usukura.jiro.w9@a.mail.nagoya-u.ac.jp.

Methods in Molecular Biology (Clifton, N.J.)

|January 31, 2025

Abstract:

Freeze-replica methods were developed in the early 1950s and have advanced through various improvements and in combination with other specimen preparation methods up to the present day. In particular, the latest immuno-freeze-etching replica of unroofed specimens is the unique method enabling three-dimensional observation of the cytoplasmic side surface structure of the plasma membrane and the membrane cytoskeleton attached to it under an electron microscope. The experimental procedures are described in detail here, listing the essential materials required for each research experiment, ranging from the classical freeze-fracture replica method to the latest unroofing immuno-freeze-etching replica method. These methods are also considered crucial for morphological approaches in the viral infection process. Indeed, in cells infected with influenza A viruses, the present methods have shown that progeny viral ribonucleoproteins (vRNPs), including the genome, are bundled by actin filaments on the cytoplasmic surface of the host cell plasma membrane.

Insights

Freeze-replica techniques offer 3D views of cell surfaces. New immuno-freeze-etching reveals viral ribonucleoproteins bundled by actin filaments in influenza A virus-infected cells.

Area of Science:

Cell Biology
Virology
Microscopy

Background:

Freeze-replica methods, developed since the 1950s, have evolved significantly.
Advanced techniques enable detailed 3D visualization of cellular structures.

Purpose of the Study:

To detail experimental procedures for classical and advanced freeze-replica methods.
To highlight the utility of immuno-freeze-etching for observing the cytoplasmic side of the plasma membrane and associated cytoskeleton.
To explore morphological aspects of viral infection processes.

Main Methods:

Classical freeze-fracture replica method.
Unroofing immuno-freeze-etching replica method for 3D observation.
Electron microscopy for high-resolution imaging.

Main Results:

The immuno-freeze-etching method allows unique 3D observation of the cytoplasmic surface of the plasma membrane and its attached cytoskeleton.
In influenza A virus-infected cells, progeny viral ribonucleoproteins (vRNPs) were observed bundled by actin filaments on the host cell plasma membrane's cytoplasmic surface.

Conclusions:

Freeze-replica methods, especially immuno-freeze-etching, are crucial for morphological studies of viral infections.
Actin filaments play a role in bundling viral ribonucleoproteins on the cytoplasmic side of the plasma membrane during influenza A virus replication.

Read full analysis

Keywords:

Electron microscopy Freeze-replica Membrane Quick freezing Unroofing Virus cell biology

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