Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.

A central feature ofSalmonellapathogenicity is the bacterium's ability to enter into non‐phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42‐ and Rac‐dependent actin cytoskeleton rearrangements. These responses are triggered by the co‐ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, aSalmonellainositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42‐dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus,Salmonellastrains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism inSalmonella‐induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P₄, a consumption of InsP₅and the activation of phospholipase C. In agreement with thein vivofindings, purified SopB specifically dephosphorylated InsP₅to Ins(1,4,5,6)P₄in vitro. Surprisingly, the inositol phosphate fluxes induced bySalmonellawere not caused exclusively by SopB. We show that the SopB‐independent inositol phosphate fluxes are the consequence of the SopE‐dependent activation of an endogenous inositol phosphatase. The ability ofSalmonellato stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.

This review assesses the authenticity of inositol hexakisphosphate (InsP(6)) being a wide-ranging regulator of many important cellular functions. Against a background in which the possible importance of localized InsP(6) metabolism is discussed, there is the facile explanation that InsP(6) is merely an "inactive" precursor for the diphosphorylated inositol phosphates. Indeed, many of the proposed cellular functions of InsP(6) cannot sustain a challenge from the implementation of a rigorous set of criteria, which are designed to avoid experimental artefacts.

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [¹³C₆]-myo-inositol or [¹³C₆]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.