The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.     

Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum

VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.     

Water and oxygen permeance of phellems isolated from trees: the role of waxes and lenticels

The outermost phellems of Abies alba Mill., Acer pseudoplatanus L., Aesculus hippocastanum L., Betula potaninii L.C. Hue and Sambucus nigra L. have been isolated enzymatically, resulting in membranes with five to seven heavily suberized cork cell layers. Water and oxygen permeances were determined for the phellem areas without lenticels. A special diaphragm made it possible to quantify permeances of single lenticels for the first time. The water permeance of phellems was in the range of 3x10(-5) to 9x10(-5) ms(-1) and can be predicted from the density of the phellem membranes with 93% accuracy. Embedded waxes amounted to 3% ( Aesculus) and up to 35% ( Betula) of the dry weight but affected water permeance only to a small degree. The sorption isotherms describing the water content of the phellems in relation to relative humidities followed a hyperbolic shape and indicated varying water contents among plant species. It is argued that water transfer across the phellems occurs via the middle lamellae. Phellem membranes were impermeable to oxygen. Removal of the waxes hardly changed this situation. Single lenticels from Betula and Sambucus were significantly more permeable to water and oxygen than phellem areas without lenticels. The water permeance was elevated by factors of 39 for Betula and 12 for Sambucus, the oxygen permeance by factors of 1,202 for Betula and 53 for Sambucus. Extraction of lenticels did not affect permeance. A quantitative comparison of the gas-exchange capacity of lenticels and stomata demonstrated the superiority of stomata. However, differences may be not more than one order of magnitude.     

Detection and relocation of cord blood nucleated red blood cells by laser scanning cytometry

BACKGROUND: Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. METHODS: CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-gamma) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. RESULTS: In CB, 59% of events double positive for Hb-gamma and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. CONCLUSIONS: Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible.     

In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation

In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.