Imaging centers as partnerships between industry and academia: NICs go global

Engel, Ulrike

doi:10.1002/biot.200900060

Cited by 1 publication

(1 citation statement)

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“…However, events at the end of the endocytic pathway take place with little synchrony over several hours, necessitating longer observation periods to capture infrequent events and making photobleaching and phototoxicity major obstacles. Only recent advances in imaging techniques [32] have allowed visualization of rapidly-moving vesicles bearing VatM-GFP at high temporal resolution and with minimal photobleaching, permitting images to be captured for up to twenty minutes at 1-second intervals. These time series have provided the first views of retrieval of the V-ATPase and have revealed multiple mechanisms by which retrieval can be accomplished in the phagocytic pathway of Dictyostelium .…”

Section: Introductionmentioning

confidence: 99%

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

et al. 2010

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BackgroundThe vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.MethodologyTo monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.Principal FindingsWe find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.Conclusions/SignficanceThus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

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Section: Introductionmentioning

confidence: 99%