Background

The midbody or the Flemming body is the central part of the intercellular bridge formed between daughter cells during the final stages of mitosis. The abscission on either side of the bridge by the endosomal sorting complexes required for transport (ESCRT) machinery, results in the physical separation of the two daughter cells (Green et al., 2012). In addition to its known function in the regulation of mitosis, recent studies have begun to elucidate post-mitotic roles for the midbody. Due to its role in the initiation of lumen formation in kidney cells, the midbody has been postulated to serve as a polarity cue (Li et al., 2014). More recently, it has been demonstrated that the midbody remnant is directly involved in primary ciliogenesis by polarised Madin-Darby canine kidney (MDCK) cells (Bernabé-Rubio et al., 2016). It has been also found to have a role in formation of the dorsoventral axis during the development of Caenorhabditis elegans (Singh and Pohl, 2014), and in defining cell fate and differentiation (Kuo et al., 2011). Previous studies have used laser ablation to impair the function of the midbody remnant. When performed in cultured cell lines, however, laser ablation can result in cell death due to damage of the plasma membrane and proximal cytosolic elements. Accordingly, we have designed a gentle procedure, which we have called ‘take-up by suction pressure’ (TUSP). TUSP allows non-deleterious midbody remnant removal from the cell surface of epithelial cells. The fundamental principle is based on using a fine-aperture glass pipette attached to patch-clamp apparatus to physically remove the midbody with applied negative pressure (Figure 1).


Figure 1. Diagram of the TUSP procedure. A. An apical intercellular bridge forms during cytokinesis in polarised epithelial cells. B. After abscission, one of the daughter cells inherits the midbody as a remnant, which will be positioned over the apical cell surface. C-E. By using a glass pipette connected to path-clamp apparatus, the midbody remnant can be removed from cells if suction pressure is applied.