Peroxisome Motility Measurement and Quantification Assay

Background

An important feature of eukaryotic cells is the presence of membrane-bound compartments (organelles), which create distinct optimised environments to promote various metabolic reactions required to sustain life. For the entire cell to function as a unit, coordination and cooperation between specialized organelles must take place. This requires a dynamic spatial organization, which allows the movement of organelles to areas of greatest metabolic need, the positioning of organelles in those areas, and the interaction with other compartments, which permits metabolic cooperation and communication amongst organelles. This is often mediated through interorganellar membrane contacts, whereby two organelles come into close apposition (Prinz, 2014; Eisenberg-Bord et al., 2016).

Peroxisomes are multifunctional organelles that play pivotal cooperative roles in the metabolism of cellular lipids and reactive oxygen species (ROS) and are thus essential for human health and development (Islinger and Schrader, 2011; Wanders et al., 2015; Waterham et al., 2016). Peroxisomes interact with many organelles involved in cellular lipid metabolism such as the endoplasmic reticulum (ER), mitochondria, lipid droplets or lysosomes (Schrader et al., 2013 and 2015). We revealed that the peroxisomal tail-anchored membrane proteins ACBD5 and ACBD4 directly interact with tail-anchored VAPB at the ER (Costello et al., 2017a; 2017b and 2017c). This interaction links both organelles together and allows transfer of lipids between them (Costello et al., 2017b; Hua et al., 2017). Whereas in plants and yeast, peroxisomes move along the actin cytoskeleton by interacting with myosin motors (Jedd and Chua, 2002; Fagarasanu et al., 2009), in mammalian cells (and filamentous fungi), peroxisomes use microtubules and dynein/kinesin motors to distribute uniformly within the cell and to reach new neighbourhoods (Schrader et al., 1996 and 2003; Guimaraes et al., 2015; Lin et al., 2016). We also found that ACBD5-VAPB interaction, which tethers peroxisomes to the ER, influences peroxisome motility (Costello et al., 2017b). Using the peroxisome motility measurement and quantification assay, we showed that loss of ACBD5, which resulted in reduced peroxisome-ER association, also increased peroxisome movement (Costello et al., 2017b). Our data indicate that organelle contact sites can modulate peroxisome (organelle) distribution and motility.

Peroxisomes are highly versatile organelles, which respond to environmental stimuli with changes in their number, size, and enzyme composition (Islinger et al., 2010). Certain stress conditions, in particular in plants, can lead to changes in the motile behaviour of peroxisomes and altered distribution (Rodríguez-Serrano et al., 2009). There is currently great interest in the measurement of peroxisome (organelle) motility and its quantification in order to understand the fundamental principles of organelle distribution, its regulation and role in organelle interaction and metabolic cooperation. Understanding these mechanisms is not just important for comprehending fundamental physiological processes but also for understanding pathogenic processes in disease etiology (Ferdinandusse et al., 2017; Yagita et al., 2017).