Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-α is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 ± 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-α induces nanoscale changes in endothelial surface architecture and that—via a tetraspanin CD9 depending mechanism—the EAPs rise above the surface to facilitate leukocyte capture

Bacteria tracking by in vivo magnetic resonance imaging

Background: Different non-invasive real-time imaging techniques have been developed over the last decades to study bacterial pathogenic mechanisms in mouse models by following infections over a time course. In vivo investigations of bacterial infections previously relied mostly on bioluminescence imaging (BLI), which is able to localize metabolically active bacteria, but provides no data on the status of the involved organs in the infected host organism. In this study we established an in vivo imaging platform by magnetic resonance imaging (MRI) for tracking bacteria in mouse models of infection to study infection biology of clinically relevant bacteria. Results: We have developed a method to label Gram-positive and Gram-negative bacteria with iron oxide nano particles and detected and pursued these with MRI. The key step for successful labeling was to manipulate the bacterial surface charge by producing electro-competent cells enabling charge interactions between the iron particles and the cell wall. Different particle sizes and coatings were tested for their ability to attach to the cell wall and possible labeling mechanisms were elaborated by comparing Gram-positive and -negative bacterial characteristics. With 5-nm citrate-coated particles an iron load of 0.015 ± 0.002 pg Fe/bacterial cell was achieved for Staphylococcus aureus. In both a subcutaneous and a systemic infection model induced by iron-labeled S. aureus bacteria, high resolution MR images allowed for bacterial tracking and provided information on the morphology of organs and the inflammatory response. Conclusion: Labeled with iron oxide particles, in vivo detection of small S. aureus colonies in infection models is feasible by MRI and provides a versatile tool to follow bacterial infections in vivo. The established cell labeling strategy can easily be transferred to other bacterial species and thus provides a conceptual advance in the field of molecular MRI.<br

Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling

Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression

Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor

Schematic model of adhesive microvilli formation.

<p>(<b>A</b>) Upon activation of endothelial cells, ICAM-1 is upregulated and processed into the plasma membrane; it associates with tetraspanin CD9 to form clusters 2-dimensional. (<b>B</b>) The clusters (or: endothelial adhesive platforms EAPs also contain JAM-A, which is not depicted here) recruit f-actin, potentially through a RhoG dependent mechanism. (<b>C</b>) The clustered adhesion platforms are propelled upwards by typically 160 ± 80 nm, thereby increasing the interaction probability with leukocytes. (<b>D</b>) Upon leukocyte contact, the microvilli are further elongated to develop a full docking structure with long filopodia engulfing the leukocyte.</p

AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation

Sketch on physiological relevance of adhesive microvilli.

<p>Geometrical considerations illustrate the impact of microvilli on binding probability of leukocytes. General assumptions are: ICAM-1 clusters (<i>adhesive platforms</i>, <i>EAP</i>) cover 10% of the endothelial surface and are equally distributed at mean distance of 2b = 2.5 μm, and leukocytes have smooth, spherical shape, radius a = 5 μm. (<b>A</b>) In a simple model of a flat endothelial surface, the chance of a leukocyte to meet an EAP upon first contact just equals the ICAM-1 surface coverage (10%). (<b>B</b>) With an arbitrary convolvement of membrane surface as in real cells (cytoskeletal roughness), many EAPs become inaccessible for leukocytes. The binding probability decreases close to zero, depending on the degree of surface roughness. (<b>C</b>) This sterical shielding effect is avoided, when EAPs are elevated above ground level. With the geometrical assumptions made above, EAPs reach a binding probability of 95% when lifted upwards by 160 nm.</p

Immunostaining of ICAM-clusters.

<p>HUVECs before or after treatment with TNF-α were subjected to fluorescent staining for f-actin using phalloidin and immunolabeling for ICAM-1 using antibodies, respectively. The focal planes of confocal laser scanning microscopy (LSM) were adjusted to the apical surface. (<b>A</b>) represents a maximum intensity projection. ICAM-1 immunoreactivity is virtually absent from control cells and the actin forms a belt at the cell periphery along the junctions. Upon activation with (<b>TNF-α</b>), f-actin forms stress fibers (<b>A</b>), which exhibit many short debranchings as seen in the zoomed image (<b>B</b>). The ICAM-1 adhesion protein forms a punctuate pattern of elongated or even triangular spots with diameter of around 1 μm (<b>A</b>) They are distributed around the perinuclear area of the cell and are often located at the debranching sites (<b>B</b>). The reconstructed side view (<b>C</b>) shows f-actin both at the basal and apical areas, and ICAM-1 only at the apical surface. (<b>D</b>) A closer view on the apical surface demonstrates the clustered appearance and colocalization of ICAM-1 and f-actin. (<b>E</b>) A highest-resolution micrograph reveals a microvilli of more than 1 μm height which are decorated with ICAM-1 (white arrows). As seen from the merged image, ICAM-1 is mostly accompanied by f-actin but not the other way round. Images shown are representative for 3 independent cell preparations. In A and B, it is maximum intensity projections, while C-E are single slices in xz-plane (x = z scale).</p