The recent identification of several novel endocytic compartments has challenged our current understanding of the topological and functional organization of the endocytic pathway. Using quantitative single vesicle imaging and acute manipulation of phosphoinositides we show that APPL endosomes, which participate in growth factor receptor trafficking and signaling, represent an early endocytic intermediate common to a subset of clathrin derived endocytic vesicles and macropinosomes. Most APPL endosomes are precursors of classical PI3P positive endosomes, and PI3P plays a critical role in promoting this conversion. Depletion of PI3P causes a striking reversion of Rab5 positive endosomes to the APPL stage, and results in enhanced growth factor signaling. These findings reveal a surprising plasticity of the early endocytic pathway. Importantly, PI3P functions as a switch to dynamically regulate maturation and signaling of APPL endosomes.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Whole-exome sequencing studies in autism spectrum disorder (ASD) have identified de novo mutations in novel candidate genes, including the synaptic gene Eighty-five Requiring 3A (EFR3A). EFR3A is a critical component of a protein complex required for the synthesis of the phosphoinositide PtdIns4P, which has a variety of functions at the neural synapse. We hypothesized that deleterious mutations in EFR3A would be significantly associated with ASD.

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained Gs signaling at endosomes. In addition to Gs, PTHR1 activates Gq signaling; yet, in contrast to the PTHR1-Gs pathway, the spatiotemporal dynamics of the Gq branch of PTHR1 signaling and its relationship to Gs signaling remain largely ill defined. Recognizing that a downstream consequence of Gq signaling is the activation of phospholipase D (PLD) enzymes, we leverage activity-based, bioorthogonal imaging tools for PLD signaling to visualize and quantify the Gq branch of PTHR1 signaling. We establish that PTHR1-Gq signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein Gq and Gs compete for ligand-bound receptors at the PM and more broadly highlight the utility of bioorthogonal tools for imaging PLDs as probes to visualize GPCR-Gq signaling.

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

Wnt signaling pathways direct key physiological decisions in development. Here, we establish a role for a pleckstrin homology domain-containing protein, PLEKHA4, as a modulator of signaling strength in Wnt-receiving cells. PLEKHA4 oligomerizes into clusters at PI(4,5)P2-rich regions of the plasma membrane and recruits the Cullin-3 (CUL3) E3 ubiquitin ligase substrate adaptor Kelch-like protein 12 (KLHL12) to these assemblies. This recruitment decreases CUL3-KLHL12-mediated polyubiquitination of Dishevelled, a central intermediate in canonical and non-canonical Wnt signaling. Knockdown of PLEKHA4 in mammalian cells demonstrates that PLEKHA4 positively regulates canonical and non-canonical Wnt signaling via these effects on the Dishevelled polyubiquitination machinery. In vivo knockout of the Drosophila melanogaster PLEKHA4 homolog, kramer, selectively affects the non-canonical, planar cell polarity (PCP) signaling pathway. We propose that PLEKHA4 tunes the sensitivities of cells toward the stimulation of Wnt or PCP signaling by sequestering a key E3 ligase adaptor controlling Dishevelled polyubiquitination within PI(4,5)P2-rich plasma membrane clusters.

Lowe syndrome and type 2 Dent disease are caused by defects in the inositol 5-phosphatase OCRL. Most missense mutations in the OCRL ASH-RhoGAP domain that are found in affected individuals abolish interactions with the endocytic adaptors APPL1 and Ses (both Ses1 and Ses2), which bind OCRL through a short phenylalanine and histidine (F&H) motif. Using X-ray crystallography, we have identified the F&H motif binding site on the RhoGAP domain of OCRL. Missense mutations associated with disease affected F&H binding indirectly by destabilizing the RhoGAP fold. By contrast, a disease-associated mutation that does not perturb F&H binding and ASH-RhoGAP stability disrupted the interaction of OCRL with Rab5. The F&H binding site of OCRL is conserved even in species that do not have an identified homolog for APPL or Ses. Our study predicts the existence of other OCRL binding partners and shows that the perturbation of OCRL interactions has a crucial role in disease.

Plasma membrane PI4P helps determine the identity of this membrane and plays a key role in signal transduction as the precursor of PI(4,5)P2 and its metabolites. Here, we report the atomic structure of the protein scaffold that is required for the plasma membrane localization and function of Stt4/PI4KIIIα, the PI 4-kinase responsible for this PI4P pool. Both proteins of the scaffold, Efr3 and YPP1/TTC7, are composed of α-helical repeats, which are arranged into a rod in Efr3 and a superhelix in Ypp1. A conserved basic patch in Efr3, which binds acidic phospholipids, anchors the complex to the plasma membrane. Stt4/PI4KIIIα is recruited by interacting with the Ypp1 C-terminal lobe, which also binds to unstructured regions in the Efr3 C terminus. Phosphorylation of this Efr3 region counteracts Ypp1 binding, thus providing a mechanism through which Stt4/PI4KIIIα recruitment, and thus a metabolic reaction of fundamental importance in cell physiology, can be regulated.

Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

Genetic defects in myelin formation and maintenance cause leukodystrophies, a group of white matter diseases whose mechanistic underpinnings are poorly understood. Hypomyelination and congenital cataract (HCC), one of these disorders, is caused by mutations in FAM126A, a gene of unknown function. We show that FAM126A, also known as hyccin, regulates the synthesis of phosphatidylinositol 4-phosphate (PtdIns(4)P), a determinant of plasma membrane identity. HCC patient fibroblasts exhibit reduced PtdIns(4)P levels. FAM126A is an intrinsic component of the plasma membrane phosphatidylinositol 4-kinase complex that comprises PI4KIIIα and its adaptors TTC7 and EFR3 (refs 5,7). A FAM126A-TTC7 co-crystal structure reveals an all-α-helical heterodimer with a large protein-protein interface and a conserved surface that may mediate binding to PI4KIIIα. Absence of FAM126A, the predominant FAM126 isoform in oligodendrocytes, destabilizes the PI4KIIIα complex in mouse brain and patient fibroblasts. We propose that HCC pathogenesis involves defects in PtdIns(4)P production in oligodendrocytes, whose specialized function requires massive plasma membrane expansion and thus generation of PtdIns(4)P and downstream phosphoinositides. Our results point to a role for FAM126A in supporting myelination, an important process in development and also following acute exacerbations in multiple sclerosis.

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.

Mutations of the inositol 5-phosphatase OCRL cause Lowe syndrome (LS), characterized by congenital cataract, low IQ, and defective kidney proximal tubule resorption. A key subset of LS mutants abolishes OCRL's interactions with endocytic adaptors containing F&H peptide motifs. Converging unbiased methods examining human peptides and the unicellular phagocytic organism Dictyostelium discoideum reveal that, like OCRL, the Dictyostelium OCRL orthologue Dd5P4 binds two proteins closely related to the F&H proteins APPL1 and Ses1/2 (also referred to as IPIP27A/B). In addition, a novel conserved F&H interactor was identified, GxcU (in Dictyostelium) and the Cdc42-GEF FGD1-related F-actin binding protein (Frabin) (in human cells). Examining these proteins in D. discoideum, we find that, like OCRL, Dd5P4 acts at well-conserved and physically distinct endocytic stations. Dd5P4 functions in coordination with F&H proteins to control membrane deformation at multiple stages of endocytosis and suppresses GxcU-mediated activity during fluid-phase micropinocytosis. We also reveal that OCRL/Dd5P4 acts at the contractile vacuole, an exocytic osmoregulatory organelle. We propose F&H peptide-containing proteins may be key modifiers of LS phenotypes.

Plasma membrane PI4P is an important direct regulator of many processes that occur at the plasma membrane and also a biosynthetic precursor of PI(4,5)P2 and its downstream metabolites. The majority of this PI4P pool is synthesized by an evolutionarily conserved complex, which has as its core the PI 4-kinase PI4KIIIα (Stt4 in yeast) and also comprises TTC7 (Ypp1 in yeast) and the peripheral plasma membrane protein EFR3. While EFR3 has been implicated in the recruitment of PI4KIIIα via TTC7, the plasma membrane protein Sfk1 was also shown to participate in this targeting and activity in yeast. Here, we identify a member of the TMEM150 family as a functional homologue of Sfk1 in mammalian cells and demonstrate a role for this protein in the homeostatic regulation of PI(4,5)P2 at the plasma membrane. We also show that the presence of TMEM150A strongly reduces the association of TTC7 with the EFR3-PI4KIIIα complex, without impairing the localization of PI4KIIIα at the plasma membrane. Collectively our results suggest a plasticity of the molecular interactions that control PI4KIIIα localization and function.

Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P₂) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIα, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIα was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIα knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P₂ because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P₂ was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIα to the definition of plasma membrane identity.

Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.

Phosphatidic acids (PAs) are glycerophospholipids that regulate key cell signaling pathways governing cell growth and proliferation, including the mTOR and Hippo pathways. Their acyl chains vary in tail length and degree of saturation, leading to marked differences in the signaling functions of different PA species. For example, in mTOR signaling, saturated forms of PA are inhibitory, whereas unsaturated forms are activating. To enable rapid control over PA signaling, we describe here the development of photoswitchable analogues of PA, termed AzoPA and dAzoPA, that contain azobenzene groups in one or both lipid tails, respectively. These photolipids enable optical control of their tail structure and can be reversibly switched between a straight trans form and a relatively bent cis form. We found that cis-dAzoPA selectively activates mTOR signaling, mimicking the bioactivity of unsaturated forms of PA. Further, in the context of Hippo signaling, whose growth-suppressing activity is blocked by PA, we found that the cis forms of both AzoPA and dAzoPA selectively inhibit this pathway. Collectively, these photoswitchable PA analogues enable optical control of mTOR and Hippo signaling, and we envision future applications of these probes to dissect the pleiotropic effects of physiological and pathological PA signaling.

The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/β-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, we examine the cellular determinants of ISC proliferation, harnessing kramer , a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. We find that Wnt signaling within Prospero-positive cells supports ISC proliferation and that kramer regulates Wnt signaling in this context by antagonizing kelch , a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/β-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.