Imaging of radiation effects on cellular 26S proteasome function in situ

Abstract

Purpose: The classical radiobiological paradigm is that DNA is the target for cell damage caused by ionising radiation. However, evidence is accumulating that other constituents, such as the membrane, organelles, and proteins, are also important targets. We have shown that the isolated 26S proteasome is one such target and here we wish to substantiate it within the cell, in situ.

Materials and methods: We used confocal microscopy to quantitatively detect and subcellularly localise radiation-induced 26S proteasome inhibition in cells expressing an ornithine decarboxylase degron that targets a fused Zoanthus species green (ZsGreen) fluorescent protein reporter specifically to the 26S proteasome.

Results: Exposure of cells to a range of radiation doses, even as low as 0.05 Gy inhibited 26S activity within minutes. Initially, punctate nuclear ZsGreen fluorescence was observed that became cytoplasmic after seven hours – a pattern distinct from the diffuse homogeneous fluorescence of cells incubated in the conventional proteasome inhibitor MG-132.

Conclusions: Our study clearly indicates that the 26S proteasome is a radiation target with physiological consequences and introduces a new perspective in mechanistic investigations of cellular responses to stresses.

Keywords:

• Proteasome
• fluorescence confocal microscopy
• ionising radiation
• ornithine decarboxylase degron-Zs-Green