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Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and cell–matrix adhesion. Central to this process is phosphatidylinositol bisphosphate (PIP2). The levels of PIP2 in the membrane are rapidly altered by the activity of phosphoinositide-directed kinases and phosphatases, and it binds to dozens of different intracellular proteins. Despite the vast literature dedicated to understanding the regulation of PIP2 in cells over past 30 years, much remains to be learned about its cellular functions. In this review, we focus on past and recent exciting results on different molecular mechanisms that regulate cellular functions by binding of specific proteins to PIP2 or by stabilizing phosphoinositide pools in different cellular compartments. Moreover, this review summarizes recent findings that implicate dysregulation of PIP2 in many diseases

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P(2) synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P(2). However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P(2), which may be fulfilled by a more general polyanionic lipid requirement.

Multicellular organisms have well-defined, tightly regulated mechanisms for cell adhesion. Heterodimeric αβ integrin receptors play central roles in this function and regulate processes for normal cell functions, including signaling, cell migration, and development, binding to the extracellular matrix, and senescence. They are involved in hemostasis and the immune response, participate in leukocyte function, and have biological implications in angiogenesis and cancer. Proper control of integrin activation for cellular communication with the external environment requires several physiological processes. Perturbation of these equilibria may lead to constitutive integrin activation that results in bleeding disorders. Furthermore, integrins play key roles in cancer progression and metastasis in which certain tumor types exhibit higher levels of various integrins. Thus, the integrin-associated signaling complex is important for cancer therapy development. During inside-out signaling, the cytoskeletal protein talin plays a key role in regulating integrin affinity whereby the talin head domain activates integrin by binding to the cytoplasmic tail of β-integrin and acidic membrane phospholipids. To understand the mechanism of integrin activation by talin, we determined the crystal structure of the talin head domain bound to the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), allowing us to design a lipid-binding–deficient talin mutant. Our confocal microscopy with talin knockout cells suggests that the talin–cell membrane interaction seems essential for focal adhesion formation and stabilization. Basal integrin activation in Chinese hamster ovary cells suggests that the lipidbinding–deficient talin mutant inhibits integrin activation. Thus, membrane attachment of talin seems necessary for integrin activation and focal adhesion formation.

Protein–lipid interactions are a key element of the function of many integral membrane proteins. These potential interactions should be considered alongside the complexity and diversity of membrane lipid composition. Inward rectifier potassium channel (Kir) Kir2.2 has multiple interactions with plasma membrane lipids: Phosphatidylinositol (4, 5)-bisphosphate (PIP₂) activates the channel; a secondary anionic lipid site has been identified, which augments the activation by PIP₂; and cholesterol inhibits the channel. Molecular dynamics simulations are used to characterize in molecular detail the protein–lipid interactions of Kir2.2 in a model of the complex plasma membrane. Kir2.2 has been simulated with multiple, functionally important lipid species. From our simulations we show that PIP₂ interacts most tightly at the crystallographic interaction sites, outcompeting other lipid species at this site. Phosphatidylserine (PS) interacts at the previously identified secondary anionic lipid interaction site, in a PIP2 concentration-dependent manner. There is interplay between these anionic lipids: PS interactions are diminished when PIP₂ is not present in the membrane, underlining the need to consider multiple lipid species when investigating protein–lipid interactions.

Inward rectifier potassium channels The regulatory lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is the primary activator of inward rectifier K + (Kir) channels. Kir channels control the resting membrane potential in a wide variety of excitable cells. The X-ray crystal structure of the Kir2.2 potassium channel in complex with a PIP 2 derivative has now been determined. One PIP 2 molecule binds to each of the four K + channel subunits near the membrane inner leaflet. On binding, a large conformational change occurs, causing the cytoplasmic domain to engage the transmembrane domain and the pore to open. This work shows the structural basis for the regulation of receptors and ion channels by lipids, an important factor in the control of cell signalling. The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells 1 , 2 . In particular, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels 2 , 3 , 4 , 5 , 6 , 7 , 8 . PIP 2 is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell’s resting membrane potential 2 , 7 , 8 , 9 . However, the molecular mechanism by which PIP 2 exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP 2 . We found that PIP 2 binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP 2 -binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP 2 binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 Å and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP 2 can control the resting membrane potential through a specific ion-channel-receptor–ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.

The interorganelle communication mediated by membrane contact sites (MCSs) is an evolutionary hallmark of eukaryotic cells. MCS connections enable the nonvesicular exchange of information between organelles and allow them to coordinate responses to changing cellular environments. In plants, the importance of MCS components in the responses to environmental stress has been widely established, but the molecular mechanisms regulating interorganelle connectivity during stress still remain opaque. In this report, we use the model plant Arabidopsis thaliana to show that ionic stress increases endoplasmic reticulum (ER)–plasma membrane (PM) connectivity by promoting the cortical expansion of synaptotagmin 1 (SYT1)-enriched ER–PM contact sites (S-EPCSs). We define differential roles for the cortical cytoskeleton in the regulation of S-EPCS dynamics and ER–PM connectivity, and we identify the accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] at the PM as a molecular signal associated with the ER–PM connectivity changes. Our study highlights the functional conservation of EPCS components and PM phosphoinositides as modulators of ER–PM connectivity in eukaryotes, and uncovers unique aspects of the spatiotemporal regulation of ER–PM connectivity in plants.

Insulin release takes place in two phases: a first rapid burst followed by a series of small exocytic bursts that coincide with pulsatile spikes in cytosolic Ca 2+ levels. The second phase is impaired in patients with type II diabetes, underscoring the importance of understanding its molecular basis. Lees et al. report a mechanism through which TMEM24, a lipid transport protein that concentrates at endoplasmic reticulum–plasma membrane contact sites, regulates the pulsatility of cytosolic Ca 2+ and phosphoinositide signaling. This process in turn regulates pulsatile insulin secretion during the slow insulin release phase. Science , this issue p. eaah6171 Direct lipid transport between the endoplasmic reticulum and the plasma membrane helps to control insulin secretion. Insulin is released by β cells in pulses regulated by calcium and phosphoinositide signaling. Here, we describe how transmembrane protein 24 (TMEM24) helps coordinate these signaling events. We showed that TMEM24 is an endoplasmic reticulum (ER)–anchored membrane protein whose reversible localization to ER-plasma membrane (PM) contacts is governed by phosphorylation and dephosphorylation in response to oscillations in cytosolic calcium. A lipid-binding module in TMEM24 transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] precursor phosphatidylinositol between bilayers, allowing replenishment of PI(4,5)P 2 hydrolyzed during signaling. In the absence of TMEM24, calcium oscillations are abolished, leading to a defect in triggered insulin release. Our findings implicate direct lipid transport between the ER and the PM in the control of insulin secretion, a process impaired in patients with type II diabetes.

ORP5 and ORP8, members of the oxysterol-binding protein (OSBP)-related proteins (ORP) family, are endoplasmic reticulum membrane proteins implicated in lipid trafficking. ORP5 and ORP8 are reported to localize to endoplasmic reticulum–plasma membrane junctions via binding to phosphatidylinositol-4-phosphate (PtdIns(4) P ), and act as a PtdIns(4) P /phosphatidylserine counter exchanger between the endoplasmic reticulum and plasma membrane. Here we provide evidence that the pleckstrin homology domain of ORP5/8 via PtdIns(4,5) P 2 , and not PtdIns(4) P binding mediates the recruitment of ORP5/8 to endoplasmic reticulum–plasma membrane contact sites. The OSBP-related domain of ORP8 can extract and transport multiple phosphoinositides in vitro, and knocking down both ORP5 and ORP8 in cells increases the plasma membrane level of PtdIns(4,5) P 2 with little effect on PtdIns(4) P . Overall, our data show, for the first time, that phosphoinositides other than PtdIns(4) P can also serve as co-exchangers for the transport of cargo lipids by ORPs.

Amyloid β (Aβ) oligomer-induced aberrant neurotransmitter release is proposed to be a crucial early event leading to synapse dysfunction in Alzheimer’s disease (AD). In the present study, we report that the release probability (Pr) at the synapse between the Schaffer collateral (SC) and CA1 pyramidal neurons is significantly reduced at an early stage in mouse models of AD with elevated Aβ production. High nanomolar synthetic oligomeric Aβ 42 also suppresses Pr at the SC-CA1 synapse in wild-type mice. This Aβ-induced suppression of Pr is mainly due to an mGluR5-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in axons. Selectively inhibiting Aβ-induced PIP 2 hydrolysis in the CA3 region of the hippocampus strongly prevents oligomeric Aβ-induced suppression of Pr at the SC-CA1 synapse and rescues synaptic and spatial learning and memory deficits in APP/PS1 mice. These results first reveal the presynaptic mGluR5-PIP 2 pathway whereby oligomeric Aβ induces early synaptic deficits in AD. The underlying mechanism of amyloid β (Aβ) oligomer-induced aberrant neurotransmitter release remains unclear. Here, authors show that the release probability at the synapse between the Schaffer collateral and CA1 pyramidal neurons is significantly reduced at an early stage in mouse models of AD with elevated Aβ production and is mainly due to an mGluR5-mediated depletion of PIP2 in axons.