Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as seeds for replication in the cytoplasm. Tau uptake is highly efficient, but only ~1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and biosensor cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.

Salmonella enters a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence.

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.

VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow–derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8–deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8–deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3–positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

Role of endogenously secreted angiotensin II in the CO2-induced stimulation of HCO3 reabsorption by renal proximal tubules

Previous studies demonstrated that the proximal tubule (PT) responds to isolated increases in basolateral ([CO2]BL) or “bath” CO2 concentration by increasing the HCO3− reabsorption rate ( J[Formula: see text]). Blockade of the rabbit apical AT1 receptor or knockout of the mouse AT1A receptor eliminates these effects, demonstrating a requirement for luminal ANG II that the PT itself synthesizes. In the present study, we examined the effects of the ACE inhibitor lisinopril on J[Formula: see text] in isolated perfused rabbit PTs (S2 segment), using out-of-equilibrium solutions to make isolated changes in [CO2]BL at a fixed baseline HCO3− concentration of 22 mM and fixed baseline pH of 7.4. Adding 60 or 240 nM lisinopril (in vitro Ki: 0.5 or 1.2 nM) to the lumen had no effect. These results are not consistent with the hypothesis that the PT secretes either angiotensinogen or ANG I. However, adding 60 nM basolateral lisinopril significantly decreased J[Formula: see text] at a [CO2]BL of 20%. Moreover, 240 nM basolateral lisinopril decreased baseline (i.e., at 5% CO2) J[Formula: see text] by one-half and completely eliminated the response to altering [CO2]BL from 0 to 20%, but left intact the stimulatory effect of 10−11 M basolateral ANG II. At extremely high concentrations (i.e., 100 μM), luminal lisinopril replicated the effects of 240 nM basolateral lisinopril. Our data are consistent with the hypothesis that lisinopril readily crosses the basolateral (but not apical) membrane to block ACE in a vesicular compartment. We conclude that the isolated PT predominantly secretes preformed ANG II, rather than angiotensinogen or ANG I.

Comparative studies of duodenal and macrophage ferroportin proteins

Intestinal epithelial cells and reticuloendothelial macrophages are, respectively, involved in diet iron absorption and heme iron recycling from senescent erythrocytes, two critical processes of iron homeostasis. These cells appear to use the same transporter, ferroportin (Slc40a1), to export iron. The aim of this study was to compare the localization, expression, and regulation of ferroportin in both duodenal and macrophage cells. Using a high-affinity purified polyclonal antibody, we analyzed the localization and expression of ferroportin protein in the spleen, liver, and duodenum isolated from normal mice as well as from well-characterized mouse models of altered iron homeostasis. Ferroportin was found to be predominantly expressed in enterocytes of the duodenum, in splenic macrophages, and in liver Kupffer cells. Interestingly, the protein species detected in these cells migrated differently on SDS-PAGE. These differences in apparent molecular masses were partly explained by posttranslational complex N-linked glycosylations. In addition, in enterocytes, the transporter was mostly expressed at the basolateral membrane, whereas in bone marrow-derived macrophages, ferroportin was found predominantly localized in the intracellular vesicular compartment. However, some microdomains positive for ferroportin were also detected at the plasma membrane of macrophages. Despite these differences, we observed a parallel upregulation of ferroportin expression in tissue macrophages and enterocytes in response to iron-restricted erythropoiesis, suggesting that iron homeostasis is likely maintained through coordinate expression of the iron exporter in both intestinal and phagocytic cells. Our data also confirm a predominant regulation of ferroportin through systemic regulator(s) likely including hepcidin.