Mixed Reality Meets Pharmaceutical Development.

As science evolves, the need for more efficient and innovative knowledge transfer capabilities becomes evident. Advances in drug discovery and delivery sciences have directly impacted the pharmaceutical industry, though the added complexities have not shortened the development process. These added complexities also make it difficult for scientists to rapidly and effectively transfer knowledge to offset the lengthened drug development timelines. While webcams, camera phones, and iPads have been explored as potential new methods of real-time information sharing, the non-"hands-free" nature and lack of viewer and observer point-of-view render them unsuitable for the R&D laboratory or manufacturing setting. As an alternative solution, the Microsoft HoloLens mixed-reality headset was evaluated as a more efficient, hands-free method of knowledge transfer and information sharing. After completing a traditional method transfer between 3 R&D sites (Rahway, NJ; West Point, PA and Schnachen, Switzerland), a retrospective analysis of efficiency gain was performed through the comparison of a mock method transfer between NJ and PA sites using the HoloLens. The results demonstrated a minimum 10-fold gain in efficiency, weighing in from a savings in time, cost, and the ability to have real-time data analysis and discussion. In addition, other use cases were evaluated involving vendor and contract research/manufacturing organizations.

Structural identification of the Vps18 ß-propeller reveals a critical role in the HOPS complex stability and function.

Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed ß-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the ß-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex.

CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion.

Protein and lipid transport along the endolysosomal system of eukaryotic cells depends on multiple fusion and fission events. Over the past few years, the molecular constituents of both fission and fusion machineries have been identified. Here, we focus on the mechanism of membrane fusion at endosomes, vacuoles and lysosomes, and in particular on the role of the two homologous tethering complexes called CORVET and HOPS. Both complexes are heterohexamers; they share four subunits, interact with Rab GTPases and soluble NSF attachment protein receptors (SNAREs) and can tether membranes. Owing to the presence of specific subunits, CORVET is a Rab5 effector complex, whereas HOPS can bind efficiently to late endosomes and lysosomes through Rab7. Based on the recently described overall structure of the HOPS complex and a number of in vivo and in vitro analyses, important insights into their function have been obtained. Here, we discuss the general function of both complexes in yeast and in metazoan cells in the context of endosomal biogenesis and maturation.

The CORVET complex promotes tethering and fusion of Rab5/Vps21-positive membranes.

Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.

Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex.

Membrane fusion within the eukaryotic endomembrane system depends on the initial recognition of Rab GTPase on transport vesicles by multisubunit tethering complexes and subsequent coupling to SNARE-mediated fusion. The conserved vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) tethering complex combines both activities. Here we present the overall structure of the fusion-active HOPS complex. Our data reveal a flexible ≈30-nm elongated seahorse-like structure, which can adopt contracted and elongated shapes. Surprisingly, both ends of the HOPS complex contain a Rab-binding subunit: Vps41 and Vps39. The large head contains in addition to Vps41 the SNARE-interacting Vps33, whereas Vps39 is found in the bulky tip of its tail. Vps11 and Vps18 connect head and tail. Our data suggest that HOPS bridges Ypt7-positive membranes and chaperones SNAREs at fusion sites.

The Rab GTPase Ypt7 is linked to retromer-mediated receptor recycling and fusion at the yeast late endosome.

Organelles of the endomembrane system need to counterbalance fission and fusion events to maintain their surface-to-volume ratio. At the late mammalian endosome, the Rab GTPase Rab7 is a major regulator of fusion, whereas the homologous yeast protein Ypt7 seems to be restricted to the vacuole surface. Here, we present evidence that Ypt7 is recruited to and acts on late endosomes, where it affects multiple trafficking reactions. We show that overexpression of Ypt7 results in expansion and massive invagination of the vacuolar membrane, which requires cycling of Ypt7 between GDP- and GTP-bound states. Invaginations are blocked by ESCRT, CORVET and retromer mutants, but not by autophagy or AP-3 mutants. We also show that Ypt7-GTP specifically binds to the retromer cargo-recognition subcomplex, which--like its cargo Vps10--is found on the vacuole upon Ypt7 overproduction. Our data suggest that Ypt7 functions at the late endosome to coordinate retromer-mediated recycling with the fusion of late endosomes with vacuoles.