Hypoxia controls plasma membrane targeting of polarity proteins by dynamic turnover of PI4P and PI(4,5)P2.

Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here, we report that hypoxia in induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins.

A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2.

SAC1 degrades its lipid substrate PtdIns4 in the endoplasmic reticulum to maintain a steep chemical gradient with donor membranes.

Gradients of PtdIns4 between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4 in a 'cis' configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in 'trans' at membrane contact sites, which could oppose lipid traffic.

Phosphoinositides and Membrane Targeting in Cell Polarity.

Selective enrichment of the polyphosphoinositides (PPIn), such as PtdIns(4,5) and PtdIns4, helps to determine the identity of the plasma membrane (PM) and regulates many aspects of cell biology through a vast number of protein effectors. Polarity proteins had long been assumed to be non-PPIn-binding proteins that mainly associate with PM/cell cortex through their extensive protein-protein interaction network. However, recent studies began to reveal that several key polarity proteins electrostatically bind to PPIn through their positively charged protein domains or structures and such PPIn-binding property is essential for their direct and specific attachment to PM.

Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis.

We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis.

Does PtdIns(4,5)P2 concentrate so it can multi-task?

Ptdns(4,5)P2 is a minor structural lipid of the plasma membrane (PM), but a master regulator of PM function. Serving either as a substrate for the generation of second messengers, or more commonly as a ligand triggering protein recruitment or activation, it regulates most aspects of PM function. Understanding how this relatively simple biological macromolecule can regulate such a vast array of different functions in parallel, is the key to understanding the biology of the PM as a whole, in both health and disease.

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay.

The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome.

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation.

A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia.

Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations.

The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action.

Polyphosphoinositide binding domains: Key to inositol lipid biology.

Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation of many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions.

A tail of new lipids.

The inositol lipids play many essential roles in eukaryotic physiology, although the action has usually focused on the special properties of their headgroup. Now, a study by Clark et al (2014) re-focuses attention on the hydrophobic lipid tails, showing that these too exhibit unique biochemical properties--and are also likely to play a fundamental role in the biology of the lipid.

A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi.

Polyphosphoinositides are an important class of lipid that recruit specific effector proteins to organelle membranes. One member, phosphatidylinositol 4-phosphate (PtdIns4P) has been localized to Golgi membranes based on the distribution of lipid binding modules from PtdIns4P effector proteins. However, these probes may be biased by additional interactions with other Golgi-specific determinants.

Distinctive changes in plasma membrane phosphoinositides underlie differential regulation of TRPV1 in nociceptive neurons.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a polymodal, Ca(2+)-permeable cation channel crucial to regulation of nociceptor responsiveness. Sensitization of TRPV1 by G-protein coupled receptor (GPCR) agonists to its endogenous activators, such as low pH and noxious heat, is a key factor in hyperalgesia during tissue injury as well as pathological pain syndromes. Conversely, chronic pharmacological activation of TRPV1 by capsaicin leads to calcium influx-induced adaptation of the channel.

Membrane biology: Making light work of lipids.

A clever genetic trick allows the lipid composition of the plasma membrane to be manipulated using light, paving the way for new investigations into the many membrane interactions that dictate cell shape, movement and communication.

Regulation of voltage-gated potassium channels by PI(4,5)P2.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences.

PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity.

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.

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu.

The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context.

Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8.

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE.

Probing the regulation of TASK potassium channels by PI4,5P₂ with switchable phosphoinositide phosphatases.

TASK channels are background K+ channels that contribute to the resting conductance in many neurons. A key feature of TASK channels is the reversible inhibition by Gq-coupled receptors, thereby mediating the dynamic regulation of neuronal activity by modulatory transmitters. The mechanism that mediates channel inhibition is not fully understood.

Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2).

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P(2). Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P.

Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information.

Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid-effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane.

Polyphosphoinositol lipids: under-PPInning synaptic function in health and disease.

Phosphoinositides (PPIn) form a unique family of lipids derived by phosphorylation of the parent compound, phosphatidylinositol. Despite being minor constituents of synaptic membranes, these lipids have exceptionally high rates of metabolic turnover and are involved with myriad aspects of pre- and post-synaptic function, from the control of the synaptic vesicle cycle to postsynaptic excitability. In this review, we outline the main synaptic processes known to be regulated by these molecules, focusing mainly but not exclusively on the major species phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells.

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis.