Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

Myelin-Associated MAL and PLP Are Unusual among Multipass Transmembrane Proteins in Preferring Ordered Membrane Domains.

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multipass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored. Here we used cell-derived giant plasma membrane vesicles (GPMVs) to systematically measure multipass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multipass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four-pass TMPs, myelin and lymphocyte protein (MAL), and proteo lipid protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterol-mediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multipass transmembrane proteins.

Distinct metaplastic and inflammatory phenotypes in autoimmune and adenocarcinoma-associated chronic atrophic gastritis.

BACKGROUND: Autoimmune gastritis (AIG) and adenocarcinoma-associated chronic atrophic gastritis (CAG) are both associated with oxyntic atrophy, but AIG patients demonstrate an increased risk of carcinoid tumors rather than the elevated risk of adenocarcinoma observed with CAG. We therefore sought to compare the characteristics of the metaplastic mucosa in AIG and CAG patients. METHODS: We examined markers for metaplasia (spasmolytic polypeptide expressing metaplasia (SPEM) and intestinal metaplasia) as well as proliferation (Ki67) and immune cell populations (neutrophils, macrophages, and eosinophils) in gastric sections from 16 female patients with autoimmune thyroiditis and AIG and 17 patients with CAG associated with gastric adenocarcinoma. RESULTS: Both AIG and CAG patients demonstrated prominent SPEM and intestinal metaplasia. However, AIG patients displayed significantly lower numbers of infiltrating macrophages and significantly reduced mucosal cell proliferation as compared to CAG patients. CONCLUSIONS: These findings indicate that, while both AIG and CAG patients display prominent oxyntic atrophy and metaplasia, the AIG patients do not show proliferative metaplastic lineages that would predispose to adenocarcinoma.

Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

BACKGROUND: The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic. RESULTS: Here, we show that while providing functional recovery, transplantation of fetal dopamine neurons into the AAV-α-synuclein rat model of PD resulted in smaller-sized grafts as compared to similar grafts placed into the 6-OHDA-lesioned striatum. Additionally, we found that cyclosporin treatment was able to promote the survival of the transplanted cells in this allografted state and surprisingly also provided therapeutic benefit in sham-operated animals. We demonstrated that delayed cyclosporin treatment afforded neurorestoration in three complementary models of PD including the Thy1-α-synuclein transgenic mouse, a novel AAV-α-synuclein mouse model, and the MPTP mouse model. We then explored the mechanisms for this benefit of cyclosporin and found it was mediated by both cell-autonomous mechanisms and non-cell autonomous mechanisms. CONCLUSION: This study provides compelling evidence in favor for the use of immunosuppression in all grafted PD patients receiving cell replacement therapy, regardless of the immunological mismatch between donor and host cells, and also suggests that cyclosporine treatment itself may act as a disease-modifying therapy in all PD patients.

Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.

Bartonella henselae is able to internalize endothelial progenitor cells (EPCs), which are resistant to the infection of other common pathogens. Bacteroides fragilis is a gram-negative anaerobe belonging to the gut microflora. It protects from experimental colitis induced by Helicobacter hepaticus through the polysaccharide A (PSA). The aim of our study was to establish: 1) whether B. fragilis colonization could protect from B. henselae infection; if this event may have beneficial effects on EPCs, vascular system and tissues. Our in vitro results establish for the first time that B. fragilis can internalize EPCs and competes with B. henselae during coinfection. We observed a marked activation of the inflammatory response by Real-time PCR and ELISA in coinfected cells compared to B. henselae-infected cells (63 vs 23 up-regulated genes), and after EPCs infection with mutant B. fragilis ΔPSA (≅90% up-regulated genes) compared to B. fragilis. Interestingly, in a mouse model of coinfection, morphological and ultrastructural analyses by hematoxylin-eosin staining and electron microscopy on murine tissues revealed that damages induced by B. henselae can be prevented in the coinfection with B. fragilis but not with its mutant B. fragilis ΔPSA. Moreover, immunohistochemistry analysis with anti-Bartonella showed that the number of positive cells per field decreased of at least 50% in the liver (20±4 vs 50±8), aorta (5±1 vs 10±2) and spleen (25±3 vs 40±6) sections of mice coinfected compared to mice infected only with B. henselae. This decrease was less evident in the coinfection with ΔPSA strain (35±6 in the liver, 5±1 in the aorta and 30±5 in the spleen). Finally, B. fragilis colonization was also able to restore the EPC decrease observed in mice infected with B. henselae (0.65 vs 0.06 media). Thus, our data establish that B. fragilis colonization is able to prevent B. henselae damages through PSA.