SNX12 Role in Endosome Membrane Transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations

Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways

Background: The small molecule Eeyarestatin I (ESI) inhibits the endoplasmic reticulum (ER)-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation) substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx) are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation. Methodology and Principal Findings: Using cytotoxicity measurements, we found no role for ESI as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain) showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking. Conclusions and Significance: ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed

Transient humoral protection against H5N1 challenge after seasonal influenza vaccination of humans.

Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing 'universal' protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain 'universal' protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines

Mechanisms of action of bnAbs map to conserved regions on HA and thereby reveal conserved vulnerabilities of influenza virus.

<p>(<b>A</b>) Influenza virus life cycle highlighting the four distinct mechanism of actions of HA head-binding (green) and stem-binding (blue) bnAb. (Panel <b>B</b>, left) X-ray structure of an uncleaved H3 trimer (A/Hong Kong/1/68 PDB 1HA0) in a space filling representation. For clarity, only one monomer of the trimer is colored (HA1 green, HA2, yellow). The head region, comprising lectin and vestigial esterase domains, and the stem region, containing the fusion machinery, are indicated with dotted black lines. The receptor binding site is plotted in blue and the cleavage site in pink. The regions around these sites (solid orange lines) are the footprints of sialic acid and trypsin, respectively. To roughly estimate the trypsin footprint, a trypsin structure (PDB 1YF4) was docked on the HA cleavage site such that the cleaved HA arginine overlapped with the bound arginine from 1YF4. HA amino-acids within 5A from trypsin were then taken as an approximation of the footprint. (Panel <b>B</b>, right) Footprints, indicated by solid cyan lines, of the bnAbs studied here superimposed on HA: CH65 and CR6261 footprints are plotted on HA from A/South Carolina/1/1918 (PDB ID 3GBN), and the CR8020 footprint on A/Hong Kong/1/1968 HA (PDB ID 3SDY). For the flu B antibodies, the B/Brisbane/60/2008 structure (PDB ID 4FQM) is used. Each of the HA structures has been colored with amino-acid conservation index, corresponding to their respective virus groups: H1 – group1, H3 – group 2 and B – entire influenza B. Conservation was calculated based on the NCBI flu database set as of December 2011, assuming a number of conservative substitutions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034-Ekiert2" target="_blank">[8]</a>. Red color corresponds to more than 99% conservation, white to less than 60% conservation. Additional human antibodies of which the epitopes and/or mechanism(s) of action are known are indicated on the far right.</p

Stem-binding bnAbs are internalized into live cells in complex with viral particles, reach late endosomes, and prevent infection.

<p>(<b>A</b>) Experimental layout. Fluorescently labeled viruses and antibodies were pre-incubated and subsequently added to live cells and tracked. Whether or not cells were eventually infected was determined by staining for influenza NP after tracking individual cells for 15 hours. (<b>B</b> and <b>C</b>) Stills of movies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s007" target="_blank">Movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s008" target="_blank">S2</a>) showing the joint and directed motion of R18-labeled A/Aichi/2/1968-X31 (H3N2) (red) and AF647-labeled CR8020 (green) (<b>B</b>), and R18-labeled A/Puerto Rico/8/1934 (H1N1) virus (red) and AF647-labeled CR6261 (green) (<b>C</b>), along <i>TubulinTracker</i>-stained microtubules (white) of live MDCK cells (nucleus, blue) approximately 30 minutes after addition of the pre-incubated virus-antibody mixtures. Dashed lines outline the trajectories of the virus-antibody complexes (red triangles) as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s007" target="_blank">movies S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s008" target="_blank">S2</a>. (<b>D</b>) A/Aichi/2/1968-X31 (H3N2) virus was pre-incubated with AF647-labeled CR8020 (green) before addition to live MDCK cells labeled with <i>LysoTracker</i> (magenta) and imaged when virus-antibody complexes reached the perinuclear region. Arrows indicate co-localization of virus-antibody complexes with low-pH vesicles (white). (<b>E</b>) As in (<b>D</b>), except that here A/Puerto Rico/8/1934 (H1N1) virus and AF647-labeled CR6261 were used. (<b>F</b>) R18-labeled A/Aichi/2/1968-X31 (H3N2) virus (red) was incubated with AF647-labeled CR8020 (green) before addition to live MDCK cells expressing a GFP-cell tracer (grey cell outline). Virus-antibody complexes (co-localization shown in yellow, compare also split channels in the inset) were detected in live cells 30 minutes after inoculation. (<b>G</b>) To determine whether internalized virus-antibody complexes prevent infection, the fate of individual cells was assessed by tracking them over night (imaged in 30 min intervals). 15 hours post-incubation (hpi) the same cells (including their progeny) were fixed and stained for expression of influenza nuclear protein (NP, blue). (<b>H</b>) Incubation of R18-labeled A/Aichi/2/1968-X31 (H3N2) virus (red) with non-binding AF647-labeled CR6261 did not result in internalization of antibody. Only viral particles were detected in live cells 30 minutes after addition of the virus-antibody mixture and infection was not prevented, as demonstrated by the expression of NP (blue) in these same cells 15 hours later (<b>I</b>). Examples of progeny cells are indicated with numbers. Scale bars B–E equal 10 µm, F–I equal 25 µm.</p

Stem-binding bnAbs prevent membrane fusion in an <i>in vitro</i> single particle fusion assay.

<p>(<b>A</b>) Assay setup in microfluidic chamber mounted on an inverted fluorescent microscope. (<b>B</b> and <b>D</b>) Stills of movies of individual R18-labeled A/Aichi/2/1968-X31 (H3N2) or (<b>C</b> and <b>E</b>) A/Puerto Rico/8/1934 (H1N1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s011" target="_blank">Movie S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080034#pone.0080034.s012" target="_blank">S6</a>) virus particles (magenta) incubated with AF488-labeled bnAbs (green) and bound to sialic acid decorated proteins embedded in a supported lipid bilayer where they co-localize (white, merge). Upon lowering the pH from 7.4 to 5.0 (t = 0 seconds), viruses incubated with only 15 nM CR8020 or CR6261 undergo HA-mediated fusion with the target membrane, visualized as a rapid increase in signal due to fluorescence dequenching followed by diffusion of R18 molecules away from the fusion site (<b>B</b> and <b>C</b>, yellow triangles), whereas no fusion events occur when viruses are incubated with 1500 nM bnAbs (<b>D</b> and <b>E</b>). Scale bars equal 3 µm; illumination conditions and image contrast settings are identical in B–E. (<b>F</b> and <b>G</b>) The percentage of H3N2 and H1N1 particles undergoing fusion after the pH drop decreases with increasing concentrations of CR8020 and CR6261, respectively (black symbols). In contrast, high concentrations of bnAbs used as non-binding control antibody have no effect on the percentage of fusion (open symbols).</p