Retromer Sets a Trap for Endosomal Cargo Sorting.

Membrane trafficking from endosomes to the trans-Golgi network or the plasma membrane is driven by the retromer complex. Through structural analysis of the cargo-bound complex, Lucas et al. describe a mechanism by which endosomal membrane recruitment and cargo recognition are integrated through cooperative interactions between retromer subunits.

Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

The roles of dynein and myosin VI motor proteins in endocytosis.

Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.

[Echocardiography as a Diagnostic Tool in Emergencies and Hemodynamic Instability]. / Rolle der Echokardiografie bei Notfällen und hämodynamischer Instabilität.

Echocardiography is an imaging method in anaesthesia and intensive care medicine which adds a new dimension to hemodynamic monitoring: the direct visualization of the cardiac function and its disruptions. The review article shows the advantages and limitations of recent transthoracic (TTE) and transesophageal (TEE) echocardiography in the diagnostic course of hemodynamic instability. For TTE and TEE focused examination techniques and sequences are illustrated with regards to their fast applicability to hemodynamic monitoring.

Self-assembled, Programmable DNA Nanodevices for Biological and Biomedical Applications.

The broad field of structural DNA nanotechnology has diverged into various areas of applications ranging from computing, photonics, synthetic biology, and biosensing to in-vivo bioimaging and therapeutic delivery, to name but a few. Though the field began to exploit DNA to build various nanoscale architectures, it has now taken a new path to diverge from structural DNA nanotechnology to functional or applied DNA nanotechnology. More recently a third sub-branch has emerged-biologically oriented DNA nanotechnology, which seeks to explore the functionalities of combinatorial DNA devices in various biological systems. In this review, we summarize the key developments in DNA nanotechnology revealing a current trend that merges the functionality of DNA devices with the specificity of biomolecules to access a range of functions in biological systems. This review seeks to provide a perspective on the evolution and biological applications of DNA nanotechnology, where the integration of DNA structures with biomolecules can now uncover phenomena of interest to biologists and biomedical scientists. Finally, we conclude with the challenges, limitations, and perspectives of DNA nanodevices in fundamental and applied research.

A sequential algorithm to detect diffusion switching along intracellular particle trajectories.

MOTIVATION: Recent advances in molecular biology and fluorescence microscopy imaging have made possible the inference of the dynamics of single molecules in living cells. Changes of dynamics can occur along a trajectory. Then, an issue is to estimate the temporal change-points that is the times at which a change of dynamics occurs. The number of points in the trajectory required to detect such changes will depend on both the magnitude and type of the motion changes. Here, the number of points per trajectory is of the order of 102, even if in practice dramatic motion changes can be detected with less points. RESULTS: We propose a non-parametric procedure based on test statistics computed on local windows along the trajectory to detect the change-points. This algorithm controls the number of false change-point detections in the case where the trajectory is fully Brownian. We also develop a strategy for aggregating the detections obtained with different window sizes so that the window size is no longer a parameter to optimize. A Monte Carlo study is proposed to demonstrate the performances of the method and also to compare the procedure to two competitive algorithms. At the end, we illustrate the efficacy of the method on real data in 2D and 3D, depicting the motion of mRNA complexes-called mRNA-binding proteins-in neuronal dendrites, Galectin-3 endocytosis and trafficking within the cell. AVAILABILITY AND IMPLEMENTATION: A user-friendly Matlab package containing examples and the code of the simulations used in the paper is available at http://serpico.rennes.inria.fr/doku.php? id=software:cpanalysis:index. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

Absolute Quantification of Drug Vector Delivery to the Cytosol.

Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.

Dissecting Oligomeric States with Photoactivated Localization Microscopy: A Numerical Model.

Although photoactivated localization microscopy offers the potential to interrogate protein interactions in the physiological environment of a cell, uncertainties in the detection efficiency of photoactivatable proteins lead to complications with data interpretation. Here, we present a numerical model that provides probabilities to detect neighboring molecules dependent on their oligomerization status, density, detection efficiency, and radius, and can be used to assess oligomeric states or detection efficiencies of two molecular species. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Extracorporeal resuscitation with carbon monoxide improves renal function by targeting inflammatory pathways in cardiac arrest in pigs.

Deleterious consequences like acute kidney injury frequently occur upon successful resuscitation from cardiac arrest. Extracorporeal life support is increasingly used to overcome high cardiac arrest mortality. Carbon monoxide (CO) is an endogenous gasotransmitter, capable of reducing renal injury. In our study, we hypothesized that addition of CO to extracorporeal resuscitation hampers severity of renal injury in a porcine model of cardiac arrest. Hypoxic cardiac arrest was induced in pigs. Animals were resuscitated using a conventional [cardiopulmonary resuscitation (CPR)], an extracorporeal (E-CPR), or a CO-assisted extracorporeal (CO-E-CPR) protocol. CO was applied using a membrane-controlled releasing system. Markers of renal injury were measured, and histopathological analyses were carried out. We investigated renal pathways involving inflammation as well as apoptotic cell death. No differences in serum neutrophil gelatinase-associated lipocalin (NGAL) were detected after CO treatment compared with Sham animals (Sham 71 ± 7 and CO-E-CPR 95 ± 6 ng/mL), while NGAL was increased in CPR and E-CPR groups (CPR 135 ± 11 and E-CPR 124 ± 5 ng/mL; P < 0.05). Evidence for histopathological damage was abrogated after CO application. CO increased renal heat shock protein 70 expression and reduced inducible cyclooxygenase 2 (CPR: 60 ± 8; E-CPR 56 ± 8; CO-E-CPR 31 ± 3 µg/mL; P < 0.05). Caspase 3 activity was decreased (CPR 1,469 ± 276; E-CPR 1,670 ± 225; CO-E-CPR 755 ± 83 pg/mL; P < 0.05). Furthermore, we found a reduction in renal inflammatory signaling upon CO treatment. Our data demonstrate improved renal function by extracorporeal CO treatment in a porcine model of cardiac arrest. CO reduced proinflammatory and proapoptotic signaling, characterizing beneficial aspects of a novel treatment option to overcome high mortality.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Internal rulers to assess fluorescent protein photoactivation efficiency.

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405 nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405 nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.

Membrane scission by the ESCRT-III complex.

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

BACKGROUND: Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. METHODS: Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. RESULTS: All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. CONCLUSIONS: For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Endocytic Roles of Glycans on Proteins and Lipids.

Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis. Types of glycans on glycoproteins and glycolipids are reviewed from the angle of their interaction with glycan-binding proteins that are at the heart of galectin lattices and GL-Lect-driven endocytosis. Examples are given to show how these mechanisms affect cellular functions ranging from cell migration and signaling to vascularization and immune modulation. Finally, outstanding challenges on the link between glycosylation and endocytosis are discussed.

A high performance liquid chromatography-tandem mass spectrometry assay for therapeutic drug monitoring of 10 drug compounds commonly used for antimicrobial therapy in plasma and serum of critically ill patients: Method optimization, validation, cross-validation and clinical application.

BACKGROUND AND AIMS: Intensive care antibiotic treatment faces challenges due to substantial pharmacokinetic differences in critically ill patients. Individualized antibiotic dosing guided by therapeutic drug monitoring (TDM) is considered to minimize the risk of treatment failure and toxicity. This study aimed to develop a valid method for simultaneous LC-MS/MS quantification of 10 drugs frequently used in intensive care antibiotic therapy for which TDM-guided dosing is recommended: piperacillin, meropenem, flucloxacillin, cefuroxime, vancomycin, colistin A and B, linezolid, ciprofloxacin and tazobactam. METHODS AND RESULTS: Thorough optimization of sample preparation and chromatography resulted in a fast and simple method based on protein precipitation of 50 µL plasma or serum and gradient elution using an Acquity UPLC HSS-T3 column. Electrospray ionization-triple quadrupole mass spectrometry in dynamic multiple reaction monitoring was used for quantification, covering the therapeutic range of each drug compound. Validation following EMA and FDA recommendations, including inter-platform validation and inter-laboratory comparison, demonstrated high accuracy, precision and robustness of the new method. The assay was successfully used to monitor plasma antibiotic levels of critically ill patients (n = 35). CONCLUSION: The established multiplex method covers major drug classes with documented dosing challenges, provides a reliable basis for the implementation of high-throughput TDM, and its application confirmed the clinical impact of TDM in a real-world setting.

Retrograde transport: two (or more) roads diverged in an endosomal tree?

Some proteins and lipids traffic from the plasma membrane to the trans Golgi network (TGN)/Golgi apparatus and the endoplasmic reticulum, via the retrograde transport route. Endosomes are an obligatory through station. Whether early, recycling and late endosomes all hand off material to the TGN have remained a matter of debate. In this review, we give a short historical overview on how retrograde transport was discovered and explored. We then summarize and critically discuss data that have been put forward in favour of the existence of trafficking interfaces between each of the different endocytic localizations and the TGN. We finally point out some conceptual and technological challenges that will have to be met to establish definite conclusions for each of these scenarios.

Inhibition of proteasomal glucocorticoid receptor degradation restores dexamethasone-mediated stabilization of the blood-brain barrier after traumatic brain injury.

OBJECTIVES: To establish the molecular background for glucocorticoid insensitivity, that is, failure to reduce edema formation and to protect blood-brain barrier integrity after acute traumatic brain injury. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male C57Bl/6N mice. INTERVENTIONS: Mechanical brain lesion by controlled cortical impact. MEASUREMENTS AND MAIN RESULTS: Our study demonstrates that 1) proteasomal glucocorticoid receptor degradation is established in brain endothelial cells after traumatic brain injury as a form of posttranslational glucocorticoid receptor modification; 2) inhibition of the proteasomal degradation pathway with bortezomib (0.2 mg/kg) in combination with the glucocorticoid dexamethasone (10 mg/kg) by subcutaneous injection 30 minutes postinjury restores levels of barrier sealing glucocorticoid receptor target occludin in brain endothelial cells, improves blood-brain barrier integrity, reduces edema formation, and limits neuronal damage after brain trauma. CONCLUSIONS: The results indicate that the stabilizing effect of glucocorticoids on the blood-brain barrier is hampered after cerebral lesions by proteasomal glucocorticoid receptor degradation in brain endothelial cells and restored by inhibition of proteasomal degradation pathways. The results provide underlying mechanisms for the clinically observed inefficacy of glucocorticoids. The novel combined treatment strategy might help to attenuate trauma-induced brain edema formation and neuronal damage as secondary effects of brain trauma.