Changes in phosphoinositide concentrations ([PI]) in cell membranes constitute important signaling events. These changes are detected via PI binding domains like the pleckstrin homology (PH) domains, e.g. PI(4,5)P₂ binding PLCd1-PH, or PH unrelated ones, like the PI(4,5)P₂ bindingtubby domain. Fluorescence labeled, these domains become a probe for the monitoring of [PI] in living cells. Since [PI] can change on a sub-second timescale, the binding kinetics of these domains might be a limiting factor in the detection of such changes. Curiously, for PLCd1-PH conflicting data on these kinetics have been reported, with dissociation time constants ranging from about 0.25 s to 3 s.

To investigate this problem, we used voltage sensing phosphatases (VSPs) to rapidly deplete PI(4,5)P₂ while monitoring the dissociation of GFP-tagged PLCd1-PH and tubby domains from the plasma membrane using total internal reflection microscopy (TIRF-M) imaging. These experiments revealed rapid dissociation of both domains. If Xl-VSP1 was used, the kinetics of PI(4,5)P₂ break down were rate limiting for the observed dissociation, whereas PI(4,5)P₂ break down by Ci-VSP or Dr-VSP was sufficiently rapid to reveal differences in the time course of dissociation between both domains, with observed time constants below 1 s.

In conclusion, we find the dissociation rates of PLCd1-PH and tubbydomains to be fast enough to faithfully track changes in [PI(4,5)P₂] on a sub-second timescale.

This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM 32/2011 MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.