Methods to measure perinuclear actin dynamics during nuclear movement in migrating cells

Abstract

The nucleus is specifically positioned within a cell in diverse biological contexts. There are multiple connections between the nuclear envelope and the cytoskeleton and these connections are involved in nuclear positioning. During cell polarization prior to cell migration, nuclear envelope proteins bind to the actin cytoskeleton and get organized into linear arrays, known as transmembrane actin-associated nuclear (TAN) lines to move the nucleus away from the leading edge. Here we describe methods to study perinuclear actin dynamics, including measurement of the thickness of actin cables coupled to TAN lines, measurement of the number of perinuclear actin cables, and ablation of perinuclear actin cables. These methods are used to identify mechanisms of nuclear positioning.