Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of Akt.

Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.

PI(5)P regulates autophagosome biogenesis.

Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles.

Two isotypes of phosphatidylinositol 3-phosphate-binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica.

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3-phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P-binding GFP-HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP-HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel-like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel-like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.

Functions of Nuclear Polyphosphoinositides.

Despite interest in phosphoinositide (PtdIns) kinases, such as PtdIns 3 kinases (PI3K), as targets for controlling plasma membrane PtdIns levels in disease, the PtdIns have another less well-known site of action in the cell nucleus.Recent studies show that PtdIns use a variety of strategies to alter DNA responses. Here, we provide an overview of these newly identified forms of gene expression control, which should be considered when studying the therapeutic use of PtdIns-directed compounds. As PI3K is one of the most important clinical targets in recent years, we will focus on two polyphosphoinositides, the PI3K substrate PtdIns(4,5)di-phosphate (PI4,5P2) and its product PtdIns(3,4,5)tri-phosphate (PI3,4,5P3).

A cascade of ER exit site assembly that is regulated by p125A and lipid signals.

The inner and outer layers of COPII mediate cargo sorting and vesicle biogenesis. Sec16A and p125A (officially known as SEC23IP) proteins interact with both layers to control coat activity, yet the steps directing functional assembly at ER exit sites (ERES) remain undefined. By using temperature blocks, we find that Sec16A is spatially segregated from p125A-COPII-coated ERES prior to ER exit at a step that required p125A. p125A used lipid signals to control ERES assembly. Within p125A, we defined a C-terminal DDHD domain found in phospholipases and PI transfer proteins that recognized PA and phosphatidylinositol phosphates in vitro and was targeted to PI4P-rich membranes in cells. A conserved central SAM domain promoted self-assembly and selective lipid recognition by the DDHD domain. A basic cluster and a hydrophobic interface in the DDHD and SAM domains, respectively, were required for p125A-mediated functional ERES assembly. Lipid recognition by the SAM-DDHD module was used to stabilize membrane association and regulate the spatial segregation of COPII from Sec16A, nucleating the coat at ERES for ER exit.

Selective phosphorylation modulates the PIP2 sensitivity of the CaM-SK channel complex.

Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins, including ion channels, through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is a key cofactor for activation of small conductance Ca2+-activated potassium channels (SKs) by Ca(2+)-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SKs. The PIP2-binding site resides at the interface of CaM and the SK C terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by casein kinase 2 reduces the affinity of PIP2 for the CaM-SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G protein-mediated hydrolysis of PIP2.

Phosphatidylinositol-3,5-bisphosphate: no longer the poor PIP2.

Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol-3,5-bisphos- phate [PtdIns(3,5)P(2) ] is a low-abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as PtdIns(4,5)P(2) , our understanding of the regulation and function of PtdIns(3,5)P(2) remained rudimentary until more recently. Here, we review many of the recent developments in PtdIns(3,5)P(2) function and regulation. PtdIns(3,5)P(2) is now known to espouse functions, not only in the regulation of endolysosome morphology, trafficking and acidification, but also in autophagy, signaling mediation in response to stresses and hormonal cues and control of membrane and ion transport. In fact, PtdIns(3,5)P(2) misregulation is now linked with several human neuropathologies including Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Given the functional versatility of PtdIns(3,5)P(2) , it is not surprising that regulation of PtdIns(3,5)P(2) metabolism is proving rather elaborate. PtdIns(3,5)P(2) synthesis and turnover are tightly coupled via a protein complex that includes the Fab1/PIKfyve lipid kinase and its antagonistic Fig4/Sac3 lipid phosphatase. Most interestingly, many PtdIns(3,5)P(2) regulators play simultaneous roles in its synthesis and turnover.

PIP3 regulates spinule formation in dendritic spines during structural long-term potentiation.

Dendritic spines are small, highly motile structures on dendritic shafts that provide flexibility to neuronal networks. Spinules are small protrusions that project from spines. The number and the length of spinules increase in response to activity including theta burst stimulation and glutamate application. However, what function spinules exert and how their formation is regulated still remains unclear. Phosphatidylinositol-3,4,5-trisphosphate (PIP3) plays important roles in cell motility such as filopodia and lamellipodia by recruiting downstream proteins such as Akt and WAVE to the membrane, respectively. Here we reveal that PIP3 regulates spinule formation during structural long-term potentiation (sLTP) of single spines in CA1 pyramidal neurons of hippocampal slices from rats. Since the local distribution of PIP3 is important to exert its functions, the subcellular distribution of PIP3 was investigated using a fluorescence lifetime-based PIP3 probe. PIP3 accumulates to a greater extent in spines than in dendritic shafts, which is regulated by the subcellular activity pattern of proteins that produce and degrade PIP3. Subspine imaging revealed that when sLTP was induced in a single spine, PIP3 accumulates in the spinule whereas PIP3 concentration in the spine decreased.

Actin dynamics is rapidly regulated by the PTEN and PIP2 signaling pathways leading to myocyte hypertrophy.

Mature cardiac myocytes are terminally differentiated, and the heart has limited capacity to replace lost myocytes. Thus adaptation of myocyte size plays an important role in the determination of cardiac function. The hypothesis tested is that regulation of the dynamic exchange of actin leads to cardiac hypertrophy. ANG II was used as a hypertrophic stimulant in mouse heart and neonatal rat ventricular myocytes (NRVMs) in culture for assessment of a mechanism for regulation of actin dynamics by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin dynamics in NRVMs rapidly increased in a PIP2-dependent manner, measured by imaging and fluorescence recovery after photobleaching (FRAP). A significant increase in PIP2 levels was found by immunoblotting in both adult mouse heart tissue and cultured NRVMs. Inhibition of phosphatase and tensin homolog (PTEN) in NRVMs markedly blunted ANG II-induced increases in actin dynamics, the PIP2 level, and cell size. Furthermore, PTEN activity was dramatically upregulated in ANG II-treated NRVMs but downregulated when PTEN inhibitors were used. The time course of the rise in the PIP2 level was inversely related to the fall in the PIP3 level, which was significant by 30 min in ANG II-treated NRVMs. However, significant translocation of PTEN to the plasma membrane occurred by 10 min, suggesting a crucial initial step for PTEN for the cellular responses to ANG II. In conclusion, PTEN and PIP2 signaling may play an important role in myocyte hypertrophy by the regulation of actin filament dynamics, which is induced by ANG II stimulation.

Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice.

Olfactory receptor neurons (ORNs), which undergo lifelong neurogenesis, have been studied extensively to understand how neurons form precise topographical networks. Neural projections from ORNs are principally guided by the genetic code, which directs projections from ORNs that express a specific odorant receptor to the corresponding glomerulus in the olfactory bulb. In addition, ORNs utilise spontaneous firing activity to establish and maintain the neural map. However, neither the process of generating this spontaneous activity nor its role as a guidance cue in the olfactory bulb is clearly understood. Utilising extracellular unit-recordings in mouse olfactory epithelium slices, we demonstrated that the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels in the somas of ORNs depolarise their membranes and boost their spontaneous firing rates by sensing basal cAMP levels; the odorant-sensitive cyclic nucleotide-gated (CNG) channels in cilia do not. The basal cAMP levels were maintained via the standing activation of ß-adrenergic receptors. Using a Tet-off system to over-express HCN4 channels resulted in the enhancement of spontaneous ORN activity and dramatically reduced both the size and number of glomeruli in the olfactory bulb. This phenotype was rescued by the administration of doxycycline. These findings suggest that cAMP plays different roles in cilia and soma and that basal cAMP levels in the soma are directly converted via HCN channels into a spontaneous firing frequency that acts as an intrinsic guidance cue for the formation of olfactory networks.

PIKfyve and its Lipid products in health and in sickness.

PIKfyve, a phosphoinositide 5-kinase synthesizing PtdIns(3,5)P2 and PtdIns5P in a cellular context, belongs to an evolutionarily ancient gene family of PtdIns(3,5)P2-synthesizing enzymes that, except for plants, are products of a single-copy gene across species. In the dozen years after its discovery, enormous progress has been made in characterizing the numerous PIKfyve cellular functions and the regulatory mechanisms that govern these functions. It became clear that PIKfyve does not act alone but, rather, it engages the scaffolding regulator ArPIKfyve and the phosphatase Sac3 to make a multiprotein "PAS" complex, so called for the first letters of the protein names. This complex relays antagonistic signals, one for synthesis, another for turnover of PtdIns(3,5)P2, whose dysregulated coordination is linked to several human diseases. The physiological significance for each protein in the PAS complex is underscored by the early lethality of the mouse models with disruption in any of the three genes. This chapter summarizes our current knowledge of the diverse and complex functionality of PIKfyve and PtdIns(3,5)P2/PtdIns5P products with particular highlights on recent discoveries of inherited or somatic mutations in PIKfyve and Sac3 linked to human disorders.

Phosphatidylinositol-3,4,5-trisphosphate regulates the formation of the basolateral plasma membrane in epithelial cells.

Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.

Role of phosphatidylinositol 3,4,5-trisphosphate in cell signaling.

Many lipids present in cellular membranes are phosphorylated as part of signaling cascades and participate in the recruitment, localization, and activation of downstream protein effectors. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is one of the most important second messengers and is capable of interacting with a variety of proteins through specific PtdIns(3,4,5)P3 binding domains. Localization and activation of these effector proteins controls a myriad of cellular functions including cell survival, proliferation, cytoskeletal rearrangement, and gene expression. Aberrations in the production and metabolism of PtdIns(3,4,5)P3 have been implicated in many human diseases including cancer, diabetes, inflammation, and heart disease. This chapter provides an overview of the role of PtdIns(3,4,5)P3 in cellular regulation and the implications of PtdIns(3,4,5)P3 dysregulation in human diseases. Additionally, recent attempts at targeting PtdIns(3,4,5)P3 signaling via small molecule inhibitors are summarized.

PtdIns(4)P signalling and recognition systems.

The Golgi apparatus is a sorting platform that exchanges extensively with the endoplasmic reticulum (ER), endosomes (Es) and plasma membrane (PM) compartments. The last compartment of the Golgi, the trans-Golgi Network (TGN) is a large complex of highly deformed membranes from which vesicles depart to their targeted organelles but also are harbored from retrograde pathways. The phosphoinositide (PI) composition of the TGN is marked by an important contingent of phosphatidylinositol-4-phosphate (PtdIns(4)P). Although this PI is present throughout the Golgi, its proportion grows along the successive cisternae and peaks at the TGN. The levels of this phospholipid are controlled by a set of kinases and phosphatases that regulate its concentrations in the Golgi and maintain a dynamic gradient that determines the cellular localization of several interacting proteins. Though not exclusive to the Golgi, the synthesis of PtdIns(4)P in other membranes is relatively marginal and has unclear consequences. The significance of PtdIns(4)P within the TGN has been demonstrated for numerous cellular events such as vesicle formation, lipid metabolism, and membrane trafficking.

PtdIns5P regulation through evolution: roles in membrane trafficking?

Phosphoinositides are lipid second messengers that are essential for many cellular processes, including signal transduction and cell compartmentalization. Among them, phosphatidylinositol 5-phosphate (PtdIns5P) is the least characterized, although several proteins involved in its regulation are implicated in human diseases. We studied the distribution of 32 PtdIns5P-metabolizing proteins in 39 eukaryotic genomes. Phylogenetic profiles identify four groups of co-evolving proteins, confirming known protein complexes and revealing new ones. The complexes comprise a phosphatase, a kinase and a regulator; this indicates that physical interactions between the three partners are necessary for the acute spatial regulation of PtdIns5P turnover. By examining PtdIns5P metabolism in this new perspective, we propose a role for PtdIns5P in membrane trafficking from late endosomal compartments to the plasma membrane.

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy.

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

Lost your orientation? Find your way with PtdIns(3,4,5)P3!

Faithful chromosome segregation requires correct positioning of the spindle during mitosis. In this issue of Developmental Cell, Toyoshima et al. describe a new mechanism for spindle orientation involving phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3]. They found that in metaphase cells, dynactin was associated with the cortex through the actin cytoskeleton, and accumulated in the midsections in a PtdIns(3,4,5)P3-dependent manner. Thus, PtdIns(3,4,5)P3 regulates spindle orientation through dynein-dynactin motor complexes.

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells.

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.

Endosome-to-cytosol transport of viral nucleocapsids.

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.

Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling.

During embryonic development, cell movement is orchestrated by a multitude of attractants and repellents. Chemoattractants applied as a gradient, such as cAMP with Dictyostelium discoideum or fMLP with neutrophils, induce the activation of phospholipase C (PLC) and phosphoinositide 3 (PI3)-kinase at the front of the cell, leading to the localized depletion of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)) and the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PI[3,4,5]P(3)). Using D. discoideum, we show that chemorepellent cAMP analogues induce localized inhibition of PLC, thereby reversing the polarity of PI(4,5)P(2). This leads to the accumulation of PI(3,4,5)P(3) at the rear of the cell, and chemotaxis occurs away from the source. We conclude that a PLC polarity switch controls the response to attractants and repellents.