Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX).

The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise. Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai ., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system.