Takanari Inoue

PI(3,4,5)P3 and PI(4,5)P2 Lipids Target Proteins with Polybasic Clusters to the Plasma Membrane

Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein–conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca2+ sensor that responds to ER Ca2+ store depletion and activates Ca2+ channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca2+ store depletion. These oligomers rapidly dissociated when ER Ca2+ stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER–PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca2+ store depletion but failed to form puncta at ER–PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 μm to reach ER–PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca2+ gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca2+, (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER–PM junctions, and (iv) activation of PM Ca2+ channels.

Rapid Chemically Induced Changes of PtdIns(4,5)P2 Gate KCNQ Ion Channels

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C–mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase–mediated signaling processes.

A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN

The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP2) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP2 and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN1–351, bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN352–403. The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP2 binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.

Synthetic Activation of Endogenous PI3K and Rac Identifies an AND -Gate Switch for Cell Polarization and Migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis [1], [2], [3], [4]. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity [5], [6], [7]. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute [8], [9], [10]. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP3) production, a polarized distribution of PIP3 was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP3 or induce cell polarization. Thus, the increase in PIP3 concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.

Robust Neuronal Symmetry Breaking by Ras-Triggered Local Positive Feedback

Neuronal polarity is initiated by a symmetry-breaking event whereby one out of multiple minor neurites undergoes rapid outgrowth and becomes the axon [1]. Axon formation is regulated by phosphatidylinositol 3-kinase (PI3K)-related signaling elements [2-10] that drive local actin [11] and microtubule reorganization [3, 12], but the upstream signaling circuit that causes symmetry breaking and guarantees the formation of a single axon is not known. Here, we use live FRET imaging in hippocampal neurons and show that the activity of the small GTPase HRas, an upstream regulator of PI3K, markedly increases in the nascent axonal growth cone upon symmetry breaking. This local increase in HRas activity results from a positive feedback loop between HRas and PI3K, locally reinforced by vesicular transport of HRas to the axonal growth cone. Recruitment of HRas to the axonal growth cone is paralleled by a decrease in HRas concentration in the remaining neurites, suggesting that competition for a limited pool of HRas guarantees that only one axon forms. Mathematical modeling demonstrates that local positive feedback between HRas and PI3K, coupled to recruitment of a limited pool of HRas, generates robust symmetry breaking and formation of a single axon in the absence of extrinsic spatial cues.

Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology.

Chemically-inducible dimerization (CID) is a powerful tool that has proved useful in solving numerous problems in cell biology and related fields. In this review, we focus on case studies where CID was able to provide insight into otherwise refractory problems. Of particular interest are the cases of lipid second messengers and small GTPases, where the “signaling paradox” (how a small pool of signaling molecules can generate a large range of responses) can be at least partly explained through results gleaned from CID experiments. We also discuss several recent technical advances that provide improved specificity in CID action, novel CID substrates that allow simultaneous orthogonal manipulation of multiple systems in one cell, and several applications that move beyond the traditional CID technique of moving a protein of interest to a specific spatiotemporal location.

A Photocleavable Rapamycin Conjugate for Spatiotemporal Control of Small GTPase Activity

We developed a novel method to spatiotemporally control the activity of signaling molecules. A newly synthesized photocaged rapamycin derivative induced rapid dimerization of FKBP (FK-506 binding protein) and FRB (FKBP-rapamycin binding protein) upon UV irradiation. With this system and the spatially confined UV irradiation, we achieved subcellularly localized activation of Rac, a member of small GTPases. Our technique offers a powerful approach to studies of dynamic intracellular signaling events.

Real-Time Measurements of Protein Dynamics Using Fluorescence Activation-Coupled Protein Labeling Method

We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.