DNase I as a probe for unpolymerized actin: revisiting a classic tool for nuclear actin research

TL;DR

This paper reviews DNase I as a tool to detect unpolymerized actin in cells, focusing on its use in nuclear actin research and practical considerations for accurate results.

Contribution

The paper revisits DNase I's molecular basis and provides practical guidance for its use in detecting unpolymerized actin in fixed cells.

Findings

DNase I binds to unpolymerized actin and can detect monomeric actin in fixed cells.

Fixation conditions strongly influence DNase I staining outcomes and signal distribution.

Divalent cations and enzymatic activity affect DNase I binding and DNA interactions.

Abstract

Actin is a key component of the cytoskeleton and also plays diverse roles within the cell nucleus. While polymerized F-actin can be detected using a wide range of probes, reliable methods to identify unpolymerized (“G-”) actin in fixed cells are relatively limited. Fluorescently labeled DNase I has long served as a high-affinity probe for monomeric actin and has recently gained renewed interest in nuclear actin research. Here, we briefly review established methods for visualizing actin in the cytoplasm and nucleus and revisit the history and molecular basis of the DNase I–actin interaction that forms the basis for DNase I staining. We then highlight practical considerations for interpreting DNase I fluorescence signals, such as its binding to filament pointed ends, the influence of divalent cations on its enzymatic activity and DNA binding, and protocol-dependent variations. Notably, fixation conditions significantly influence staining outcomes, affecting the distribution of nuclear versus cytoplasmic signals. Overall, this review aims to provide a concise framework for effectively using DNase I staining to monitor cellular pools of unpolymerized actin in a consistent and interpretable way.

The online version contains supplementary material available at 10.1007/s00424-026-03158-z.

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