PI P2 Research Articles

Specialized contact sites regulate the fusion of chlamydial inclusion membranes

The intracellular bacterial pathogen Chlamydia trachomatis replicates within a membrane-bound compartment called the inclusion. Upon infection with several chlamydiae, each bacterium creates its own inclusion, resulting in multiple inclusions within each host cell. Ultimately, these inclusions fuse together in a process that requires the chlamydial protein IncA. Here, we show that inclusions form unique contact sites (inclusion contact sites, ICSs) prior to fusion, that serve as fusogenic platforms in which specific lipids and chlamydial proteins concentrate. Fusion depends on IncA clustering within ICSs and is regulated by PI(3,4)P2 and sphingolipids. As IncA concentrates within ICSs, its C-terminus likely interacts in trans with IncA on the apposing membrane, securing a high concentration of IncA at fusion sites. This regulatory mechanism contrasts with eukaryotic or viral fusion systems that are either composed of multiple proteins or use a change in pH to initiate membrane fusion. Thus, our study demonstrates that Chlamydia-mediated membrane fusion is primarily regulated by specific structural domains in IncA and its local organization on the inclusion membrane, which is affected by the host cell lipid composition.

Prokaryotic expression, purification, and activity of the inositol polyphosphate 5-phosphatase Gs5PTase8 from wild soybean

Inositol polyphosphate-5-phosphatase (5PTase) is a key enzyme in the inositol signaling pathway. It hydrolyzes the 5-phosphate on the inositol ring of inositol phosphate (IP) or phosphatidylinositol phosphate (PIP). However, there is limited reports on the homologous genes in soybean. This study cloned the salt tolerant gene Gs5PTase8 from wild soybean (Glycine soja S. & Z.) and explored its substrate. Gs5PTase8 encodes 493 amino acid residues. The sequence alignment and phylogenetic tree showed that this gene was conserved in plants. RT-qPCR was employed to determine the expression of Gs5PTase8 in different tissues of soybean and the results showed that Gs5PTase8 was mainly expressed in soybean roots. To investigate the hydrolytic substrates, we constructed pET28a-Gs5PTase8 and pGEX4T1-Gs5PTase8 for the Escherichia coli expression system and only obtained the recombinant protein GST-Gs5PTase8. The induction conditions for the protein expression including the isopropyl beta-d-thiogalactopyranoside (IPTG) concentration and temperature (16 ℃, 30 ℃, and 37 ℃) were optimized. The expression level was highest when the expression was induced overnight with 0.2 mmol/L IPTG at 16 ℃. The SDS-PAGE results showed that the recombinant protein had a relative molecular weight of 75 kDa and presented a single band after purification, with the purity reaching over 95%. The yield of the recombinant protein determined by the BCA method was 4.9 mg/L LB. The hydrolytic substrates of this enzyme in vitro included IP3 [inositol(1, 4, 5)trisphosphate], IP4 [inositol(1, 3, 4, 5)tetrakisphosphate], PI(4, 5)P2 [phosphatidylinositol(4, 5) bisphosphate] and PI(3, 4, 5)P3 [phosphatidylinositol(3, 4, 5)trisphosphate]. This study provides a scientific basis for further research on the molecular mechanism of Gs5PTase8 involved in salt tolerance.

TRPML1 gating modulation by allosteric mutations and lipids

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

Lysosomal activity depends on TRPML1-mediated Ca2+ release coupled to incoming vesicle fusions

The lysosomal cation channel TRPML1/MCOLN1 facilitates autophagic degradation during amino acid starvation based on studies involving long-term TRMPL1 modulation. Here we show that lysosomal activation (more acidic pH and higher hydrolase activity) depends on incoming vesicle fusions. We identify an immediate, calcium-dependent role of TRPML1 in lysosomal activation through promoting autophagosome-lysosome fusions and lysosome acidification within 10-20 minutes of its pharmacological activation. Lysosomes also become more fusion competent upon TRPML1 activation via increased transport of lysosomal SNARE proteins syntaxin 7 and VAMP7 by SNARE carrier vesicles. We find that incoming vesicle fusion is a prerequisite for lysosomal Ca2+ efflux that leads to acidification and hydrolytic enzyme activation. Physiologically, the first vesicle fusions likely trigger generation of the phospholipid PI(3,5)P2 that activates TRPML1, and allosteric TRPML1 activation in the absence of PI(3,5)P2 restores autophagosome-lysosome fusion and rescues abnormal SNARE sequestration within lysosomes. We thus identify a prompt role of TRPML1-mediated calcium signaling in lysosomal fusions, activation, and SNARE trafficking.

The Assembly of HTLV-1-How Does It Differ from HIV-1?

Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag-PM interactions and CA assembly.

Ebola Virus Matrix Protein VP40 Single Mutations G198R and G201R Significantly Enhance Plasma Membrane Localization.

Viral proteins frequently undergo single or multiple amino acid mutations during replication, which can significantly alter their functionality. The Ebola virus matrix protein VP40 is multifunctional but primarily responsible for creating the viral envelope by binding to the inner leaflet of the host cell plasma membrane (PM). Changes to the VP40 surface cationic charge via mutations can influence PM interactions, resulting in altered viral assembly and budding. A recent mutagenesis study evaluated the effects of several mutations and found that mutations G198R and G201R enhanced VP40 assembly at the PM and virus-like particle budding. These two mutations lie in the loop region of the C-terminal domain (CTD), which directly interacts with the PM. To understand the role of these mutations in PM localization at the molecular level, we performed both all-atom and coarse-grained molecular dynamics simulations using a dimer-dimer configuration of VP40, which contains the CTD-CTD interface. Our studies indicate that the location of mutations on the outer surface of the CTD regions can lead to changes in membrane binding orientation and degree of membrane penetration. Direct PI(4,5)P2 interactions with the mutated residues seem to further stabilize and pull VP40 into the PM, thereby enhancing interactions with numerous amino acids that were otherwise infrequently or completely inaccessible. These multiscale computational studies provide new insights at the atomic and molecular level as to how VP40-PM interactions are altered through single amino acid mutations. Given the high case fatality rates associated with Ebola virus disease in humans, it is essential to explore the mechanisms of viral assembly in the presence of mutations to mitigate the severity of the disease and understand the potential of future outbreaks.

Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy.

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.

Abstract C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C026.

TRPML1 gating modulation by allosteric mutations and lipids.

Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.

New insights into the regulation and roles of phosphatidylinositol 3,4-bisphosphate.

Phosphoinositides (PIPs) are phospholipids and components of the cellular membrane. In mammals, seven phosphorylated derivatives of PIPs have been identified. Among them, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] is produced by lipid phosphatases (e.g., SHIP2) or by lipid kinases PI3KC2α and PI3KC2β. Although PI(3,4)P2 is undetectable in normal mouse or human tissues and common cell lines, it appears in a mouse prostate cancer model and in cells exposed to oxidative stress, indicating that PI(3,4)P2 is involved in the pathogenesis of some diseases. Here, I summarize recent findings on the cellular roles and pathophysiological significance of PI(3,4)P2.

Differential functions of phosphatidylinositol 4-kinases in neurotransmission and synaptic development.

Phosphoinositides, such as PI(4,5)P2, are known to function as structural components of membranes, signalling molecules, markers of membrane identity, mediators of protein recruitment and regulators of neurotransmission and synaptic development. Phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P, which are precursors for PI(4,5)P2, but may also have independent functions. The roles of PI4Ks in neurotransmission and synaptic development have not been studied in detail. Previous studies on PI4KII and PI4KIIIβ at the Drosophila larval neuromuscular junction have suggested that PI4KII and PI4KIIIβ enzymes may serve redundant roles, where single PI4K mutants yielded mild or no synaptic phenotypes. However, the precise synaptic functions (neurotransmission and synaptic growth) of these PI4Ks have not been thoroughly studied. Here, we used PI4KII and PI4KIIIβ null mutants and presynaptic-specific knockdowns of these PI4Ks to investigate their roles in neurotransmission and synaptic growth. We found that PI4KII and PI4KIIIβ appear to have non-overlapping functions. Specifically, glial PI4KII functions to restrain synaptic growth, whereas presynaptic PI4KIIIβ promotes synaptic growth. Furthermore, loss of PI4KIIIβ or presynaptic PI4KII impairs neurotransmission. The data presented in this study uncover new roles for PI4K enzymes in neurotransmission and synaptic growth.

The scaffolding protein IQGAP1 enhances EGFR signaling by promoting oligomerization and preventing degradation

IQGAP1 is a large, multi-domain scaffold that connects and modulates different signaling networks including the one initiated by epidermal growth factor (EGF). In this study, we have used live cell fluorescence imaging methods along with other biochemical techniques to follow the mechanisms used by IQGAP1 to enhance EGF signaling. We show that IQGAP1 enhances EGF signaling by promoting the oligomerization of its receptor, EGFR, upon EGF addition along with concurrent IQGAP oligomerization. Using cellular markers, we find that IQGAP1 promotes the plasma membrane localization of EGFR and promotes association to one of its phosphoinositide lipid pathway ligands, PI(3,4,5)P3. Additionally, we find that binding of EGFR to IQGAP1 protects EGFR from lysosomal degradation. Taken together, our results show that IQGAP1 enhances EGF-mediated pathway progression through mechanisms that augment simple scaffolding activities.

Stimulation of skeletal muscle angiogenesis in aged rats by (+)-epicatechin: Identification of underlying mechanisms

Studies have linked the development of sarcopenia to reductions in capillary vessel network density and suggest that such changes may partly account for the loss of skeletal muscle (SkM) mass and function. Thus, the interest in identifying and evaluating safe and effective candidate compounds that can stimulate angiogenesis. We previously reported on the capacity of (+)-epicatechin (+Epi) to reverse SkM atrophy and loss of function in aged rats. We also reported on the apparent angiogenic effect of +Epi in mouse brain cortex. However, no studies have evaluated the angiogenic potential of +Epi in SkM. Using 23 month old male rats, we evaluated the angiogenic effect of 8 weeks of 1 mg/kg/day oral treatment of +Epi on SkM (gastrocnemius). To identify underlying mechanisms, we evaluated the PI3K/eNOS pathway, as well as the expression of the angiogenic factors HIF1-α, VEGF and VEGFR2. Results demonstrate that + Epi stimulates increases in protein levels for HIF1-α (∼35%), VEGF (∼40%), VEGFR2 (∼25%), as well as CD31 (capillaries, ∼25%). Such effects were linked with the activation (phosphorylation) of PI3K (∼30%), eNOS (∼90%), VEGFR2 at T951 (∼90%) and T996 amino acid sites (∼110%). +Epi also stimulated increased synthesis of phosphatidylinositol trisphosphate (∼30%) and tetrahydrobiopterine (∼90%), as well as of nitric oxide (∼140% in gastrocnemius and ∼70% in plasma) and cyclic guanosine monophosphate (∼90%) in gastrocnemius and in plasma (∼40%). In summary, in aged rats oral treatment with +Epi stimulates SkM angiogenesis and such actions may partly account for the reversal of the deleterious effects of sarcopenia.

HIV-1 Gag Polyprotein Affinity to the Lipid Membrane Is Independent of Its Surface Charge.

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

Understanding P-Rex regulation: structural breakthroughs and emerging perspectives.

Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gβγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis.

Endocannabinoid regulation of inward rectifier potassium (Kir) channels.

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The functions of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. Using computational and Surface plasmon resonance analysis, endocannabinoid regulation of Kir2.1 channels appears to be the result of altered membrane properties, rather than through direct protein-lipid interactions. Furthermore, differences in endocannabinoid effects on Kir4.1 and Kir7.1 channels, indicating that endocannabinoid regulation is not conserved among Kir family members. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells.

Phosphatidylinositol-3-phosphate mediates Arc capsid secretion through the multivesicular body pathway

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalize with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knockdown, cells in mouse cortical neurons reduces their surface GlutA1 levels. These results suggest that unlike the HIV Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), the assembly of Arc capsids is mediated by PI3P at endocytic membranes. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane’s inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

Parkinson’s disease gene, Synaptojanin1, dysregulates the surface maintenance of the dopamine transporter

Missense mutations of PARK20/SYNJ1 (synaptojanin1/Synj1) were found in complex forms of familial Parkinsonism. However, the Synj1-regulated molecular and cellular changes associated with dopaminergic dysfunction remain unknown. We now report a fast depletion of evoked dopamine and impaired maintenance of the axonal dopamine transporter (DAT) in the Synj1 haploinsufficient (Synj1+/−) neurons. While Synj1 has been traditionally known to facilitate the endocytosis of synaptic vesicles, we provide in vitro and in vivo evidence demonstrating that Synj1 haploinsufficiency results in an increase of total DAT but a reduction of the surface DAT. Synj1+/− neurons exhibit maladaptive DAT trafficking, which could contribute to the altered DA release. We showed that the loss of surface DAT is associated with the impaired 5’-phosphatase activity and the hyperactive PI(4,5)P2–PKCβ pathway downstream of Synj1 deficiency. Thus, our findings provided important mechanistic insight for Synj1-regulated DAT trafficking integral to dysfunctional DA signaling, which might be relevant to early Parkinsonism.

Thermal stability of bivalent cation/phosphoinositide domains in model membranes

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.