Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5+ stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5+ compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5+ phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.—Lippuner, C., Paape, D., Paterou, A., Brand, J., Richardson, M., Smith, A. J., Hoffmann, K., Brinkmann, V., Blackburn, C., Aebischer, T. Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice

Ein Stomatin-Dimer moduliert die Aktivierung von säure-abhängigen Ionenkanälen

Stomatin proteins oligomerize at membranes and have been implicated in ion channel regulation and membrane trafficking. To obtain mechanistic insights into their function, I determined three crystal structures of the conserved stomatin domain of mouse stomatin that assembles into a banana-shaped dimer. I could show that this dimerization is crucial for the modulation of acid- sensing ion channel 3 (ASIC3) and ASIC2a activities. A hydrophobic pocket at the inside of the concave surface is open in the presence of an internal peptide ligand and closes in the absence of this ligand, and we demonstrate a function of this pocket in the inhibition of ASIC3 and ASIC2a activity. In one crystal form, stomatin assembles via two conserved surfaces into a cylindrical oligomer, and these oligomerisation surfaces are also essential for the repression of ASIC3-mediated currents. The assembly mode of stomatin uncovered in this study might serve as a model to understand oligomerisation processes of related membrane-remodelling proteins, such as flotillin and prohibitin.Stomatin-verandte Proteine assemblieren an der Zellmembranen und spielen eine wichtige Rolle in der Regulation von Ionenkanälen. Um detailierte Aussagen über die Funktion dieser Protein zu erhalten wurden mittels Kristallröntgenbeugungsanalyse drei Strukturen vom Maus Stomatin gelöst. Die Analyse der Strukturen zeigte, das Stomatin als Dimer vorliegt, der eine bananenförmige Form hat. Im weiteren konnte gezeigt werden, dass diese Dimerisierung eine wichtige Rolle für die Regulierung der Ionen Kanäle ASIC3 und ASIC2a spielt. Eine hydrophobe Tasche in der Innenseite der konkaven Oberfläche des Stomatin Dimers ist geöffnet in der Anwesenheit eines kurzen internen Peptidliganden und schliesst sich, sobald dieser mutiert wird. Dass diese Tasche wichtig für die Funktion von Stomatin ist, konnte durch Mutagenese gezeigt werden. In einer der drei Kristallformen wurden Interaktionsflächen identifiziert, die zu einer zylindrischen Anordnung der Stomatin Dimere führt. Mutagenesen in diesen Interaktionaflächen verhindern die Modulation des ASIC3 Kanals. Dieser Aufbau der Stomatin Oligomers könnte einen wichtigen Beitrag zum Verständnis der Zusammensetzung von anderen SPFH Proteinen, wie z. B. Flotillin oder Prohibitin liefern