Cell-particles interaction - selective uptake and transport of microdiamonds.

Diamond particles have recently emerged as novel agents in cellular studies because of their superb biocompatibility. Their unique characteristics, including small size and the presence of fluorescent color centers, stimulate many important applications. However, the mechanism of interaction between cells and diamond particles-uptake, transport, and final localization within cells-is not yet fully understood. Herein, we show a novel, to the best of our knowledge, cell behavior wherein cells actively target and uptake diamond particles rather than latex beads from their surroundings, followed by their active transport within cells. Furthermore, we demonstrate that myosin-X is involved in cell-particle interaction, while myosin-II does not participate in particle uptake and transport. These results can have important implications for drug delivery and improve sensing methods that use diamond particles.

Organization of two kinesins in a two-dimensional microtubule network.

In intracellular active transport, molecular motors are responsible for moving biological cargo along networks of microtubules that serve as scaffolds. Cargo dynamics can be modified by different features of microtubule networks such as geometry, density, orientation modifications. Also, the dynamical behaviour of the molecular motors is determined by the microtubule network and by the individual and/or collective action of the motors. For example, unlike single kinesins, the mechanistic behavior of multiple kinesins varies from one experiment to another. However, the reasons for this experimental variability are unknown. Here we show theoretically how non-radial and quasi-radial microtubule architectures modify the collective behavior of two kinesins attached on a cargo. We found out under which structural conditions transport is most efficient and the most likely way in which kinesins are organized in active transport. In addition, with motor activity, mean intermotor distance and motor organization, we determined the character of the collective interaction of the kinesins during transport. Our results demonstrate that two-dimensional microtubule structures promote branching due to crossovers that alter directionality in cargo movement and may provide insight into the collective organization of the motors. Our article offers a perspective to analyze how the two-dimensional network can modify the cargo-motor dynamics for the case in which multiple motors move in different directions as in the case of kinesin and dynein.

Ion and lipid orchestration of secondary active transport.

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

An active transport dual adaptive nanocarrier designed to overcome the corneal microenvironment for neovascularization therapy.

The eyes have a complicated microenvironment with many clearance mechanisms, making it challenging for effective drug delivery to the targeted areas of the eyes. Substrate transport mediated by active transporters is an important way to change drug metabolism in the ocular microenvironment. We designed multifunctional, dual-adaptive nanomicelles (GSCQ@NTB) which could overcome multiple physiological barriers by acting on both the efflux transporter and influx transporter to achieve deep delivery of the P-gp substrate in the cornea. Specifically, an effective "triple" antiangiogenic agent, nintedanib (NTB), was loaded into the biocompatible micelles. The expression of the efflux transporter was reversed by grafting quercetin. The peptide (glycylsarcosine, GS) was modified to target the influx transporter "Peptide Transporter-1" (PepT-1). Quercetin (QRT) and nintedanib (NTB) were transported to the cornea cooperatively, achieving long retention on the ocular surface and high compatibility. In a New Zealand rabbit model, within 8 hours after local administration, GSCQ@NTB was enriched in corneal stromal neovascularization and effectively inhibited the progress of neovascularization. Its effectiveness is slightly better than that in the first-line clinical application of steroids. In this study, we introduce the preparation of a dual adaptive nano-micelle system, which may provide an effective non-invasive treatment for corneal neovascularization.

YTHDC1-Modified m6A Methylation of Hsa_circ_0102678 Promotes Keratinocyte Inflammation Induced by Cutibacterium acnes Biofilm through Regulating miR-146a/TRAF6 and IRAK1 Axis.

INTRODUCTION: CircRNAs are closely related to many human diseases; however, their role in acne remains unclear. This study aimed to determine the role of hsa_circ_0102678 in regulating inflammation of acne. METHODS: First, microarray analysis was performed to study the expression of circRNAs in acne. Subsequently, RNase R digestion assay and fluorescence in situ hybridization assay were utilized to confirm the characteristics of hsa_circ_0102678. Finally, qRT-PCR, Western blotting analysis, immunoprecipitation, luciferase reporter assay, circRNA probe pull-down assay, biotin-labeled miRNA pull-down assay, RNA immunoprecipitation assay, and m6A dot blot assay were utilized to reveal the functional roles of hsa_circ_0102678 on inflammation induced by C. acnes biofilm in human primary keratinocytes. RESULTS: Our investigations showed that the expression of hsa_circ_0102678 was significantly decreased in acne tissues, and hsa_circ_0102678 was a type of circRNAs, which was mainly localized in the cytoplasm of primary human keratinocytes. Moreover, hsa_circ_0102678 remarkably affected the expression of IL-8, IL-6, and TNF-α, which induced by C. acnes biofilm. Importantly, mechanistic studies indicated that the YTHDC1 could bind directly to hsa_circ_0102678 and promote the export of N6-methyladenosine-modified hsa_circ_0102678 to the cytoplasm. Besides, hsa_circ_0102678 could bind to miR-146a and sponge miR-146a to promote the expression of IRAK1 and TRAF6. CONCLUSION: Our findings revealed a previously unknown process by which hsa_circ_0102678 promoted keratinocyte inflammation induced by C. acnes biofilm via regulating miR-146a/TRAF6 and IRAK1 axis.

The ArsQ permease and transport of the antibiotic arsinothricin.

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

Protocol and Software for Automated Detection of Lysosome Active "Runs" and "Flights" with Wavelet Transform Approach.

Lysosomes are highly dynamic degradation/recycling organelles that harbor sophisticated molecular sensors and signal transduction machinery through which they control cell adaptation to environmental cues and nutrients. The movements of these signaling hubs comprise persistent, directional runs-active, ATP-dependent transport along the microtubule tracks-interspersed by short, passive movements and pauses imposed by cytoplasmic constraints. The trajectories of individual lysosomes are usually obtained by time-lapse imaging of the acidic organelles labeled with LysoTracker dyes or fluorescently-tagged lysosomal-associated membrane proteins LAMP1 and LAMP2. Subsequent particle tracking generates large data sets comprising thousands of lysosome trajectories and hundreds of thousands of data points. Analyzing such data sets requires unbiased, automated methods to handle large data sets while capturing the temporal heterogeneity of lysosome trajectory data. This chapter describes integrated and largely automated workflow from live cell imaging to lysosome trajectories to computing the parameters of lysosome dynamics. We describe an open-source code for implementing the continuous wavelet transform (CWT) to distinguish trajectory segments corresponding to active transport (i.e., "runs" and "flights") versus passive lysosome movements. Complementary cumulative distribution functions (CDFs) of the "runs/flights" are generated, and Akaike weight comparisons with several competing models (lognormal, power law, truncated power law, stretched exponential, exponential) are performed automatically. Such high-throughput analyses yield useful aggregate/ensemble metrics for lysosome active transport.

Active transport nanochelators for the reduction of liver iron burden in iron overload.

Liver dysfunction and failure account for a major portion of premature deaths in patients suffering from various iron associated pathogeneses, particularly primary and secondary iron overload disorders, despite intensive treatment. The liver is a central player in iron homeostasis and a major iron storage organ, and currently, there are no active approaches for the excretion of excess liver iron. Herein, we report a new method for the rapid reduction of iron burden in iron overload diseases by developing a new class of liver targeted nanochelators with favorable pharmacokinetics and biodistribution. The new nanochelators bypass the reticuloendothelial system and specifically target hepatocytes without non-specific accumulation in other organs. The targeted nanochelators bound and neutralized excess iron in the liver and from the vasculature and, eventually leading to rapid hepatobiliary excretion of labile iron. Further, these rapidly excreted nanochelators did not induce toxicity in the liver, were highly cytocompatible in both iron overload and non-loaded conditions, and were promising in mitigating iron triggered free radical oxidative damage. These studies provide key insights into the development of organ targeted nanochelating systems and the rapid reduction of iron burden in vivo. This methodology allows for further development of nanotherapeutics for specific iron overload diseases.

Gordura corporal, índice de massa corporal e intensidade da atividade física de acordo com transporte ativo e a prática esportiva em escolares. Um estudo transversal / Body fat, body mass index and physical activity intensity according to active transport and sports practice in schoolchildren. A cross-sectional study

Contexto: A obesidade infantil ocasiona diversas doenças e uma das formas para combatê-la é a atividade física, que exerce um papel fundamental. Objetivo: Comparar as diferentes intensidades da atividade física mensurada objetivamente de acordo com o transporte ativo, a prática de esportes e as atividades físicas estruturadas e seu impacto na gordura corporal e índice de massa corporal (IMC) em escolares. Desenho e local: Estudo transversal de amostra por critério de conveniência, realizado em São Caetano do Sul pelo Centro de Estudos do Laboratório de Aptidão Física de São Caetano do Sul (CELAFISCS). Métodos: Foram avaliadas um total de 584 crianças (277 meninos) que atenderam aos critérios de inclusão. A amostra foi dividida em grupos segundo o transporte (ativo e passivo) e a prática esportiva (sim e não). Para análise estatística foi utilizado o teste t Student e o teste U de Mann-Whitney. Para o ajuste das variáveis foi utilizada a análise de covariância (ANCOVA). Resultados: Os meninos demonstraram que, independentemente do tempo de transporte, há efeito do tipo do transporte sobre a atividade física (AF) durante a semana, de intensidade moderada, moderada-vigorosa, AF durante o final de semana de intensidade moderada, moderada-vigorosa e vigorosa. As meninas demonstraram efeito do tipo de transporte sobre a AF durante a semana na AF de intensidade moderada e de intensidade moderada-vigorosa. A gordura corporal e o IMC não apresentaram diferenças entre os grupos. As práticas esportivas não tiveram diferenças significativas em nenhuma das variáveis. Conclusões: O transporte ativo atingiu os níveis de intensidade moderada, moderada-vigorosa durante a semana, tanto no masculino como no feminino. No final de semana, além dessas, a intensidade vigorosa foi encontrada nos meninos.

Understanding Mechanisms of Secondary Active Transport by Analyzing the Effects of Mutations and Stoichiometry.

Biological cells frequently exhibit a so-called secondary active transport by moving various species across their membranes. In this mode of transport, an energetically favorable transmembrane gradient of one type of molecule is used to drive another type of molecule in the energetically unfavorable direction against their gradient. Although it is well established that conformational transitions play a critical role in functioning of transporters, the molecular details of underlying mechanisms remain not well understood. Here, we utilize a recently developed theoretical method to understand better the microscopic picture of secondary active transport. Specifically, we evaluate how mutations in different parts of transporters affect their dynamic properties. In addition, we present a possible explanation on existence of different stoichiometries in the secondary active transport. Our theoretical analysis clarifies several important aspects of complex biological transport phenomena.

Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Theoretical study of active secondary transport: Unexpected differences in molecular mechanisms for antiporters and symporters.

Successful functioning of biological cells relies on efficient translocation of different materials across cellular membranes. An important part of this transportation system is membrane channels that are known as antiporters and symporters. They exploit the energy stored as a trans-membrane gradient of one type of molecules to transport the other types of molecules against their gradients. For symporters, the directions of both fluxes for driving and driven species coincide, while for antiporters, the fluxes move in opposite directions. There are surprising experimental observations that despite differing only by the direction of transport fluxes, the molecular mechanisms of translocation adopted by antiporters and symporters seem to be drastically different. We present chemical-kinetic models to quantitatively investigate this phenomenon. Our theoretical approach allows us to explain why antiporters mostly utilize a single-site transportation when only one molecule of any type might be associated with the channel. At the same time, the transport in symporters requires two molecules of different types to be simultaneously associated with the channel. In addition, we investigate the kinetic constraints and efficiency of symporters and compare them with the same properties of antiporters. Our theoretical analysis clarifies some important physical-chemical features of cellular trans-membrane transport.

Effects of defective motors on the active transport in biosensors powered by biomolecular motors.

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport in living cells. They were proposed to drive molecular shuttles for the active transport of analytes, thus significantly accelerating the sensing process of biosensors. Integrating motor proteins into biosensors requires their immobilisation on the operating surfaces. However, this process makes some motor proteins defective, slowing analyte detection. Here, we investigated the movements of molecular shuttles on surfaces in the presence of active and defective motors using a Brownian dynamics simulation, and elucidated the effects of defective motor proteins on the transport efficiency of the shuttles. We found that the motility of shuttles depends on the fraction of active motors relative to defective ones and that over 90% of the surface-bound motor proteins must remain active for efficient transport. The high fraction of active motors required for efficient transport can be attributed to the difference in the binding lifetimes of active and defective motors to shuttles. These results provide insights into how motors accumulate on sensor surfaces and set a guideline for the choice of polymer materials for biosensors powered by motor proteins.

Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate.

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition.

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug-drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.

Tenapanor: A new treatment option for hyperphosphatemia in end stage kidney disease.

PURPOSE: This narrative review explores the currently published studies that have evaluated tenapanor for the treatment of hyperphosphatemia in end-stage kidney disease (ESKD) patients on hemodialysis. This medication's new phosphate lowering mechanism of action reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux by inhibition of the sodium/hydrogen exporter isoform 3 (NHE3). Tenapanor additionally prevents active transcellular phosphate absorption compensation by decreasing the expression of sodium phosphorus 2b transport protein (NaPi2b). METHODS: A comprehensive search of the literature was conducted using PubMed and ClinicalTrials.gov search engines. The search term "tenapanor hyperphosphatemia" was used for study retrieval. Results were limited to studies published in the English language and excluded review articles. Human, animal, and in vitro studies were included. No date range was specified. RESULTS: A total of 11 primary studies were identified and included in this review, the largest human study of which enrolled 236 patients. Each study is presented in table format along with measured end points. CONCLUSIONS: Tenapanor is the first drug in its class that lowers hyperphosphatemia in ESKD patients through a novel mechanism of action involving paracellular inactive transport. Although more studies are needed, early results indicate that tenapanor may have a place in managing hyperphosphatemia in ESKD patients both as monotherapy and as an adjunct to existing phosphate binder therapy.

Dihydroartemisinin improves hypercholesterolemia in ovariectomized mice via enhancing vectorial transport of cholesterol and bile acids from blood to bile.

The increase of concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the serum of postmenopausal women is the important risk factor of the high morbidity of cardiovascular diseases of old women worldwide. To test the anti-hypercholesterolemia function of dihydroartemisinin (DHA) in postmenopausal women, ovariectomized (OVX) mice were generated, and DHA were administrated to OVX mice for 4 weeks. The blood and liver tissues were collected for biochemical and histological tests respectively. The mRNA and protein expression levels of genes related to metabolism and transport of cholesterol, bile acid and fatty acid in the liver or ileum were checked through qPCR and western blot. DHA could significantly reduce the high concentrations of TC and LDL-C in the serum and the lipid accumulation in the liver of ovariectomized mice. The expression of ABCG5/8 was reduced in liver of OVX mice, and DHA could up-regulate the expression of them. Genes of transport proteins for bile salt transport from blood to bile, including Slc10a1, Slco1b2 and Abcb11, were also significantly up-regulated by DHA. DHA also down-regulated the expression of Slc10a2 in the ileum of OVX mice to reduce the absorption of bile salts. Genes required for fatty acid synthesis and uptake, such as Fasn and CD36, were reduced in the liver of OVX mice, and DHA administration could significantly up-regulate the expression of them. These results demonstrated that DHA could improve hypercholesterolemia in OVX mice through enhancing the vectorial transport of cholesterol and bile acid from blood to bile.

An assessment of the transport mechanism and intraneuronal stability of L-carnosine.

L-Carnosine (ß-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H+-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 µM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10-4 nmol/mg (prot)/min (with 119 µM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.

Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane.

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter.

Capillary endothelial cells of the human blood-brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.