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 of Salmonella pathogenicity 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, a Salmonella inositol 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, Salmonella strains 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 in Salmonella‐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 InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were 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 of Salmonella to 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.

Edited by Alex TokerPhosphate has multiple functions that direct the survival of all living organisms: in its organic form, P i is a component of genomic material, it serves as an energy currency, and it is ubiquitous in cell signaling. Thus, P i homeostasis is essential to life, but the mechanisms by which this occurs in humans and other metazoans are largely unknown (1, 2). Most of the previous work in this field of research has focused on yeast models (3-5). In particular, recent studies with Saccharomyces cerevisiae have revealed a new function in P i homeostasis for inositol pyrophosphates (5). The latter are soluble, intracellular signals that contain multiple phosphates and diphosphates; up to seven (InsP 7 ) 4 or eight (InsP 8 ) phosphates in total are crammed around a six-carbon inositol ring (see Refs. 6 -8 and Fig. 1). In S. cerevisiae, levels of one inositol pyrophosphate, 5-InsP 7 , track perturbations to P i homeostasis (5).This P i -sensing activity of 5-InsP 7 appears to reflect it being synthesized by a kinase class (kcs1 in yeast; IP6Ks in metazoans) that exhibits an unusually low affinity for ATP (9, 10). Consequently, cellular levels of 5-InsP 7 in yeast decrease in response to the drop in [ATP] that accompanies extracellular [P i ] depletion (5, 11). Furthermore, these ATP-driven changes in 5-InsP 7 levels appear to comprise a dynamic signaling response because 5-InsP 7 regulates proteins that maintain P i homeostasis through interactions with their SPX domains (5). However, it is not known to what extent this signaling response is applicable to metazoan cells, which lack orthologs of many of the yeast genes that function in P i sensing and P i homeostasis (2).