Amphetamine Induces Sex-Dependent Loss of the Striatal Dopamine Transporter in Sensitized Mice.

Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph administration induces transient DAT endocytosis, which, among other Amph effects on dopaminergic neurons, elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT are unknown. Hence, we developed a 14 d Amph-sensitization protocol in knock-in mice expressing HA-epitope-tagged DAT (HA-DAT) and investigated the effects of Amph challenge on sensitized HA-DAT animals. The Amph challenge resulted in the highest locomotor activity on Day 14 in both sexes, which was sustained for 1 h in male but not female mice. Strikingly, significant (by 30-60%) loss of the HA-DAT protein in the striatum was caused by the Amph challenge of sensitized males but not females. Amph also reduced V max of dopamine transport in the striatal synaptosomes of males without changing K m values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT colocalization with the endosomal protein VPS35 only in Amph-challenged males. Amph-induced loss of striatal HA-DAT in sensitized mice was blocked by chloroquine, vacuolin-1, and inhibitor of Rho-associated kinases ROCK1/2, indicative of the involvement of endocytic trafficking in the DAT protein loss. Interestingly, an apparent degradation of HA-DAT protein was observed in the nucleus accumbens and not in the dorsal striatum. We propose that Amph challenge in sensitized mice triggers Rho-mediated endocytosis and post-endocytic trafficking of DAT in a brain-region-specific and sex-dependent manner.

Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Endocytic down-regulation of the striatal dopamine transporter by amphetamine in sensitized mice in sex-dependent manner.

Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph is proposed to cause transient DAT endocytosis which among other Amph effects on dopaminergic neurons elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT traffic are unknown. Hence, we developed a 14-day Amph-sensitization protocol in knock-in mice expressing HA-epitope tagged DAT (HA-DAT) and investigated effects of Amph challenge on HA-DAT in sensitized animals. Amph challenge resulted in the highest locomotor activity on day 14 in both sexes, which was however sustained for 1 hour in male but not female mice. Strikingly, significant (by 30-60%) reduction in the amount of the HA-DAT protein in striatum was observed in response to Amph challenge of sensitized males but not females. Amph reduced Vmax of dopamine transport in striatal synaptosomes of males without changing Km values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT co-localization with the endosomal protein VPS35 only in males. Amph-induced HA-DAT down-regulation in the striatum of sensitized mice was blocked by chloroquine, vacuolin-1 (inhibitor of PIKfive kinase), and inhibitor of Rho-associated kinases (ROCK1/2), indicative of the involvement of endocytic trafficking in DAT down-regulation. Interestingly, HA-DAT protein down-regulation was observed in nucleus accumbens and not in dorsal striatum. We propose that Amph challenge in sensitized mice leads to ROCK-dependent endocytosis and post-endocytic traffic of DAT in a brain-region-specific and sex-dependent manner.

The Alkylamine Stimulant 1,3-Dimethylamylamine Exhibits Substrate-Like Regulation of Dopamine Transporter Function and Localization.

The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used nonmedically as an appetite suppressant and exercise performance enhancer despite adverse cardiovascular effects that have limited its legal status. There is scant research describing the mechanism of action of DMAA, making it difficult to gauge risks or therapeutic potential. An important molecular target of structurally related phenethylamines, such as amphetamine, for regulating mood, cognition, movement, and the development of substance use disorder is the dopamine transporter, which limits the range and magnitude of dopamine signaling via reuptake from the extracellular space. The present studies were therefore initiated to characterize the effects of DMAA on dopamine transporter function. Specifically, we tested the hypothesis that DMAA exhibits substrate-like effects on dopamine transporter function and trafficking. In transport assays in human embryonic kidney cells, DMAA inhibited dopamine uptake by the human dopamine transporter in a competitive manner. Docking analysis and molecular dynamics simulations supported these findings, revealing that DMAA binds to the S1 substrate binding site and induces a conformational change from outward-facing open to outward-facing closed states, similar to the known substrates. Further supporting substrate-like effects of DMAA, the drug stimulated dopamine transporter endocytosis in a heterologous expression system via cocaine- and protein kinase A-sensitive mechanisms, mirroring findings with amphetamine. Together, these data indicate that DMAA elicits neurologic effects by binding to and regulating function of the dopamine transporter. Furthermore, pharmacologic distinctions from amphetamine reveal structural determinants for regulating transporter conformation and add mechanistic insight for the regulation of dopamine transporter endocytosis. SIGNIFICANCE STATEMENT: The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used as an appetite suppressant and athletic performance enhancer and is structurally similar to amphetamine, but there is scant research describing its mechanism of action. Characterizing the effects of DMAA on dopamine transporter function supports evaluation of potential risks and therapeutic potential while also revealing mechanistic details of dynamic transporter-substrate interactions.

CRISPR/Cas9 Gene Editing of HeLa Cells to Tag Proteins with mNeonGreen.

Subcellular localization dynamics of proteins involved in signal transduction processes is crucial in determining the signaling outcome. However, there is very limited information about the localization of endogenous signaling proteins in living cells. For example, biochemical mechanisms underlying the signaling pathway from epidermal growth factor (EGF) receptor (EGFR) to RAS-RAF and ERK1/2/MAPK are well understood, whereas the operational domains of this pathway in the cell remain poorly characterized. Tagging of endogenous components of signaling pathways with fluorescent proteins allows more accurate characterization of their intracellular dynamics at their native expression levels controlled by endogenous regulatory mechanisms, thus avoiding possible tainting effects of overexpression and mistargeting. In this study, we describe methodological approaches to label components of the EGFR-RAS-MAPK pathway, such as Grb2, KRAS, and NRAS, with the fluorescent protein mNeonGreen (mNG) using CRISPR/Cas9 gene-editing, as well as generation of homozygous single-cell clones of the edited target protein.

Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR.

Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.

EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions.

The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS-containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.

Placental response to maternal SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic affected people at all ages. Whereas pregnant women seemed to have a worse course of disease than age-matched non-pregnant women, the risk of feto-placental infection is low. Using a cohort of 66 COVID-19-positive women in late pregnancy, we correlated clinical parameters with disease severity, placental histopathology, and the expression of viral entry and Interferon-induced transmembrane (IFITM) antiviral transcripts. All newborns were negative for SARS-CoV-2. None of the demographic parameters or placental histopathological characteristics were associated with disease severity. The fetal-maternal transfer ratio for IgG against the N or S viral proteins was commonly less than one, as recently reported. We found that the expression level of placental ACE2, but not TMPRSS2 or Furin, was higher in women with severe COVID-19. Placental expression of IFITM1 and IFITM3, which have been implicated in antiviral response, was higher in participants with severe disease. We also showed that IFITM3 protein expression, which localized to early and late endosomes, was enhanced in severe COVID-19. Our data suggest an association between disease severity and placental SARS-CoV-2 processing and antiviral pathways, implying a role for these proteins in placental response to SARS-CoV-2.

Mechanism of p38 MAPK-induced EGFR endocytosis and its crosstalk with ligand-induced pathways.

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.

Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had a massive impact on human lives worldwide. While the airborne SARS-CoV-2 primarily affects the lungs, viremia is not uncommon. As placental trophoblasts are directly bathed in maternal blood, they are vulnerable to SARS-CoV-2. Intriguingly, the human fetus is largely spared from SARS-CoV-2 infection. We tested whether the human placenta expresses the main SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin and showed that ACE2 and TMPRSS2 are expressed in the trophoblast rather than in other placental villous cells. While furin is expressed in the main placental villous cell types, we surveyed, trophoblasts exhibit the highest expression. In line with the expression of these entry factors, we demonstrated that a SARS-CoV-2 pseudovirus could enter primary human trophoblasts. Mechanisms underlying placental defense against SARS-CoV-2 infection likely involve postentry processing, which may be germane for mitigating interventions against SARS-CoV-2.IMPORTANCE Pregnant women worldwide have been affected by COVID-19. As the virus is commonly spread to various organs via the bloodstream and because human placental trophoblasts are directly bathed in maternal blood, feto-placental infection by SARS-CoV-2 seems likely. However, despite the heightened risk to pregnant women, thus far the transmission risk of COVID-19 to the feto-placental unit seems extremely low. This has been recently attributed to a negligible expression of SARS-CoV-2 entry factors in the human placenta. We therefore sought to explore the expression of the entry factors ACE2 and TMPRSS2 in the different cell types of human placental villi. Using a combination of transcriptome sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), in situ hybridization, and immunofluorescence, we found that trophoblasts, but not the other main villous cell types, express ACE2 and TMPRSS2, with a broad expression of furin. Correspondingly, we also showed that primary human trophoblasts are permissive to entry of SARS-CoV-2 pseudovirus particles.

Mechanisms for Regulating and Organizing Receptor Signaling by Endocytosis.

Intricate relationships between endocytosis and cellular signaling, first recognized nearly 40 years ago through the study of tyrosine kinase growth factor receptors, are now known to exist for multiple receptor classes and to affect myriad physiological and developmental processes. This review summarizes our present understanding of how endocytosis orchestrates cellular signaling networks, with an emphasis on mechanistic underpinnings and focusing on two receptor classes-tyrosine kinase and G protein-coupled receptors-that have been investigated in particular detail. Together, these examples provide a useful survey of the current consensus, uncertainties, and controversies in this rapidly advancing area of cell biology.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity.

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule-induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Trophoblastic extracellular vesicles and viruses: Friends or foes?

Cells produce cytoplasmic vesicles to facilitate the processing and transport of RNAs, proteins, and other signaling molecules among intracellular organelles. Moreover, most cells release a range of extracellular vesicles (EVs) that mediate intercellular communication in both physiological and pathological settings. In addition to a better understanding of their biological functions, the diagnostic and therapeutic prospects of EVs, particularly the nano-sized small EVs (sEVs, exosomes), are currently being rigorously pursued. While EVs and viruses such as retroviruses might have evolved independently, they share a number of similar characteristics, including biogenesis pathways, size distribution, cargo, and cell-targeting mechanisms. The interplay of EVs with viruses has profound effects on viral replication and infectivity. Our research indicates that sEVs, produced by primary human trophoblasts, can endow other non-placental cell types with antiviral response. Better insights into the interaction of EVs with viruses may illuminate new ways to attenuate viral infections during pregnancy, and perhaps develop new antiviral therapeutics to protect the feto-placental unit during critical times of human development.

Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments.

Dopamine transporter (DAT) controls dopamine neurotransmission by clearing synaptically released dopamine. However, trafficking itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this "long-range" trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of ∼20 d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed.SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is a key regulator of dopamine neurotransmission and a target of abused psychostimulants. In the present study, we examined, for the first time, mechanisms of the long-range traffic of DAT in intact brain and acute brain slices from the knock-in mouse expressing epitope-tagged DAT. Using a combination of confocal, super-resolution and EM, we defined DAT localization and its membrane diffusion parameters in medial forebrain bundle axonal tracts connecting midbrain somatodendritic and striatal axonal compartments of dopaminergic neurons. In contrast to the widely accepted model of long-range axonal transport, our studies suggest that DAT traffics between midbrain and striatum, mainly by lateral diffusion in the plasma membrane with only a limited contribution of vesicular transport in recycling endosomes.

Placental small extracellular vesicles: Current questions and investigative opportunities.

The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.

Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.

Endocytosis: A Turnover Mechanism Controlling Ion Channel Function.

Ion channels (IChs) are transmembrane proteins that selectively drive ions across membranes. The function of IChs partially relies on their abundance and proper location in the cell, fine-tuned by the delicate balance between secretory, endocytic, and degradative pathways. The disruption of this balance is associated with several diseases, such as Liddle's and long QT syndromes. Because of the vital role of these proteins in human health and disease, knowledge of ICh turnover is essential. Clathrin-dependent and -independent mechanisms have been the primary mechanisms identified with ICh endocytosis and degradation. Several molecular determinants recognized by the cellular internalization machinery have been discovered. Moreover, specific conditions can trigger the endocytosis of many IChs, such as the activation of certain receptors, hypokalemia, and some drugs. Ligand-dependent receptor activation primarily results in the posttranslational modification of IChs and the recruitment of important mediators, such as ß-arrestins and ubiquitin ligases. However, endocytosis is not a final fate. Once internalized into endosomes, IChs are either sorted to lysosomes for degradation or recycled back to the plasma membrane. Rab proteins are crucial participants during these turnover steps. In this review, we describe the major ICh endocytic pathways, the signaling inputs triggering ICh internalization, and the key mediators of this essential cellular process.

Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations.

Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis.

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells.

Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.