It's what's inside that counts

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) at cellular membranes can recruit and activate various proteins that activate downstream signalling pathways. However, little is known about the dynamics of PtdIns(3,4,5)P₃ production, because there has been no technique to analyse the spatial and temporal dynamics of this process quantitatively in single living cells. However, now, in Nature Cell Biology, Umezawa and colleagues present a new fluorescent indicator — called 'fllip' — that has allowed them to study PtdIns(3,4,5)P₃ dynamics in vivo based on fluorescence resonance energy transfer (FRET).

Fllip contains a pleckstrin-homology (PH) domain (which binds PtdIns(3,4,5)P₃ selectively) fused between cyan- and yellow-fluorescent-protein (CFP and YFP) variants using rigid linkers. One of these linkers contains a di-glycine motif that functions as a hinge, and an additional rigid linker attaches the indicator to a membrane-localization sequence (MLS). When the central PH domain of fllip binds PtdIns(3,4,5)P₃, a 'flip-flop-type' conformational change occurs through the flexible hinge, which can be detected as intramolecular FRET from CFP to YFP.

After showing that the PtdIns(3,4,5)P₃-binding specificity of the PH domain was unaffected by its inclusion in fllip, Umezawa and co-workers showed that fllip could be targeted to specific membranes using specific MLSs. Fllip that was observed mainly at the plasma membrane was designated fllip-pm, whereas fllip that could be seen at endomembranes (that is, at the endoplasmic reticulum (ER) and Golgi) was designated fllip-em. Furthermore, they showed that the PtdIns(3,4,5)P₃-dependent increase in FRET between CFP and YFP is dose dependent.

Next, Umezawa and colleagues studied the response of fllip-pm and fllip-em to physiological stimulation by studying the effect of platelet-derived growth factor (PDGF) on PDGF receptor (PDGFR)-expressing cells. PDGF promotes PDGFR dimerization, which results in PDGFR phosphorylation and activation. Phosphorylated PDGFR then recruits and activates phosphatidylinositol 3-kinase (PI3K), which results in PtdIns(3,4,5)P₃ production.

The authors found that, after PDGF stimulation, the levels of PtdIns(3,4,5)P₃ increased at the plasma membrane as expected, and also at endomembranes. There was, however, a delay before the increase occurred at endomembranes. In addition, the increase at endomembranes was two- to threefold greater than at the plasma membrane. So, what is the molecular mechanism behind the PtdIns(3,4,5)P₃ increase at endomembranes?

Umezawa and co-workers showed that overexpression of a dominant-negative dynamin mutant blocked the PDGF-induced increase in PtdIns(3,4,5)P₃ levels at endomembranes, but not at the plasma membrane (dynamin controls clathrin-mediated endocytosis of receptor tyrosines kinases (RTKs) such as PDGFR). They observed the same effect when they overexpressed protein tyrosine phosphatase-1B (this phosphatase is localized exclusively to the cytoplasmic surface of the ER, where it has been proposed to inactivate RTKs). These results indicate that PtdIns(3,4,5)P₃ production “...is increased at endomembranes when activated PDGFR is internalized from the plasma membrane to endomembranes by clathrin-coated endocytosis, thereby activating [PI3K] at endomembranes”.

So, PtdIns(3,4,5)P₃ at endomembranes is produced in situ, and is not transported to this location by plasma-membrane-derived endocytic vesicles. Umezawa and colleagues have therefore suggested that endocytosed RTKs activate PI3K on endomembranes before they are downregulated at the ER. This work has addressed a long-standing question about how PtdIns(3,4,5)P₃ activates its downstream signalling pathways at intracellular compartments that are remote from the plasma membrane, and has also provided us with a new type of fluorescent indicator that could be used to study other lipid second messengers.