Localization of the tubby domain, a PI(4,5)P2 biosensor, to E-Syt3-rich endoplasmic reticulum-plasma membrane junctions.

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs.

S-acylation anchors remorin proteins to the plasma membrane but does not primarily determine their localization in membrane microdomains.

Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.

The intrinsically disordered N-terminal region of AtREM1.3 remorin protein mediates protein-protein interactions.

The longstanding structure-function paradigm, which states that a protein only serves a biological function in a structured state, had to be substantially revised with the description of intrinsic disorder in proteins. Intrinsically disordered regions that undergo a stimulus-dependent disorder-to-order transition are common to a large number of signaling proteins. However, little is known about the functionality of intrinsically disordered regions in plant proteins. Here we investigated intrinsic disorder in a plant-specific remorin protein that has been described as a signaling component in plant-microbe interactions. Using bioinformatic, biochemical, and biophysical approaches, we characterized the highly abundant remorin AtREM1.3, showing that its N-terminal region is intrinsically disordered. Although only the AtREM1.3 C-terminal domain is essential for stable homo-oligomerization, the N-terminal region facilitates this interaction. Furthermore, we confirmed the stable interaction between AtREM1.3 and four isoforms of the importin α protein family in a yeast two-hybrid system and by an in planta bimolecular fluorescent complementation assay. Phosphorylation of Ser-66 in the intrinsically disordered N-terminal region decreases the interaction strength with the importin α proteins. Hence, the N-terminal region may constitute a regulatory domain, stabilizing these interactions.

Two cooperative binding sites sensitize PI(4,5)P2 recognition by the tubby domain.

Phosphoinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. The dominant PI of the plasma membrane is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. One of only two PI(4,5)P2 recognition domains characterized in detail is the tubby domain. It is essential for targeting proteins into cilia involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here, we used coarse-grained molecular dynamics simulations to explore PI(4,5)P2 binding by the prototypic tubby domain. The comparatively low PI(4,5)P2 affinity of the previously described canonical binding site is underpinned in a cooperative manner by a previously unknown, adjacent second binding site. Mutations in the previously unknown site impaired PI(4,5)P2-dependent plasma membrane localization in living cells and PI(4,5)P2 interaction in silico, emphasizing its importance for PI(4,5)P2 affinity. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of tubby-like proteins that underlies delivery of ciliary cargo.

The N-terminal homology (ENTH) domain of Epsin 1 is a sensitive reporter of physiological PI(4,5)P2 dynamics.

Phospholipase Cß (PLCß)-induced depletion of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) transduces a plethora of signals into cellular responses. Importance and diversity of PI(4,5)P2-dependent processes led to strong need for biosensors of physiological PI(4,5)P2 dynamics applicable in live-cell experiments. Membrane PI(4,5)P2 can be monitored with fluorescently-labelled phosphoinositide (PI) binding domains that associate to the membrane depending on PI(4,5)P2 levels. The pleckstrin homology domain of PLCδ1 (PLCδ1-PH) and the C-terminus of tubby protein (tubbyCT) are two such sensors widely used to study PI(4,5)P2 signaling. However, certain limitations apply to both: PLCδ1-PH binds cytoplasmic inositol-1,4,5-trisphosphate (IP3) produced from PI(4,5)P2 through PLCß, and tubbyCT responses do not faithfully report on PLCß-dependent PI(4,5)P2 dynamics. In searching for an improved biosensor, we fused N-terminal homology domain of Epsin1 (ENTH) to GFP and examined use of this construct as genetically-encoded biosensor for PI(4,5)P2 dynamics in living cells. We utilized recombinant tools to manipulate PI or Gq protein-coupled receptors (GqPCR) to stimulate PLCß signaling and characterized PI binding properties of ENTH-GFP with total internal reflection (TIRF) and confocal microscopy. ENTH-GFP specifically recognized membrane PI(4,5)P2 without interacting with IP3, as demonstrated by dialysis of cells with the messenger through a patch pipette. Utilizing Ci-VSP to titrate PI(4,5)P2 levels, we found that ENTH-GFP had low PI(4,5)P2 affinity. Accordingly, ENTH-GFP was highly sensitive to PLCß-dependent PI(4,5)P2 depletion, and in contrast to PLCδ1-PH, overexpression of ENTH-GFP did not attenuate GqPCR signaling. Taken together, ENTH-GFP detects minute changes of PI(4,5)P2 levels and provides an important complementation of experimentally useful reporters of PI(4,5)P2 dynamics in physiological pathways.

A method to control phosphoinositides and to analyze PTEN function in living cells using voltage sensitive phosphatases.

Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P2 and PI(3,4,5)P3 levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P3. Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P2 and PI(3,4,5)P3 levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.

Ion channel regulation by phosphoinositides analyzed with VSPs-PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility.

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K(+) channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs.

Potent and specific bactericidal effect of juglone (5-hydroxy-1,4-naphthoquinone) on the fire blight pathogen Erwinia amylovora.

A screening of plant quinones for inhibiting effects on the bacterial fire blight pathogen Erwinia amylovora was performed. The most active compound, juglone from walnuts, has a potent and specific bactericidal effect on E. amylovora and minimal inhibitory concentrations of only 2.5-10 µM, with stronger effects at lower, but still physiological, pH values. In vitro tests with juglone and inoculated flowers of apple (Malus domestica) showed an efficacy of 67% in preventing infection. In two years of field tests juglone had variable degrees of efficacy ranging from 40 to 82%, seemingly due to environmental conditions. A phytotoxic reaction to juglone, which is known for its allelopathic effect on plants, was restricted to browning of petals; later fruit russeting was not observed. Juglone is a promising candidate for the development of a new environmentally friendly plant protectant to replace the antibiotic streptomycin currently used in fire blight control.