Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL).

Methods And Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

Aims: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown.

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus.

The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses.

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that-like SynCAM 1-MPP2 is situated at the periphery of the postsynaptic density (PSD).

Simultaneous Multicolor DNA-PAINT without Sequential Fluid Exchange Using Spectral Demixing.

Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach "DNA-PAINT" robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by "Exchange-PAINT", which requires sequential exchange of the imaging solution and thus leads to extended acquisition times.

A cytosolic disulfide bridge-supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A.

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability.

The ARFRP1-dependent Golgi scaffolding protein GOPC is required for insulin secretion from pancreatic β-cells.

Objective: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood.

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl /H exchange, we now generate Clcn3 mice in which ClC-3 is converted into a Cl channel.

RIM-BP2 primes synaptic vesicles recruitment of Munc13-1 at hippocampal mossy fiber synapses.

All synapses require fusion-competent vesicles and coordinated Ca-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca-secretion coupling.

Vasopressin Increases Urinary Acidification V1a Receptors in Collecting Duct Intercalated Cells.

Background: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function.

Methods: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys.

The cell adhesion protein CAR is a negative regulator of synaptic transmission.

The Coxsackievirus and adenovirus receptor (CAR) is essential for normal electrical conductance in the heart, but its role in the postnatal brain is largely unknown. Using brain specific CAR knockout mice (KO), we discovered an unexpected role of CAR in neuronal communication. This includes increased basic synaptic transmission at hippocampal Schaffer collaterals, resistance to fatigue, and enhanced long-term potentiation.

A SEPT1-based scaffold is required for Golgi integrity and function.

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi.

Beclin1-driven autophagy modulates the inflammatory response of microglia via NLRP3.

Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia-mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia.

Vesicular Synaptobrevin/VAMP2 Levels Guarded by AP180 Control Efficient Neurotransmission.

Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis.

Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins.

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton.

Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

Background: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.

Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation.

During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl-) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl-/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl- conductor.