Endo-Lysosomal Vesicles Positive for Rab7 and LAMP1 Are Terminal Vesicles for the Transport of Dextran

Humphries, William H.; Szymanski, Craig; Payne, Christine K.

doi:10.1371/journal.pone.0026626

Cited by 107 publications

(94 citation statements)

References 44 publications

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“…LAMP1, a membrane glycoprotein found on LE and LYs, arrived shortly after this conversion. Vacuoles positive for both Rab7a and LAMP1 are known to serve as terminal organelles during trafficking of the fluid phase marker dextran 60. The change in Rabs and other markers thus suggested that VACV‐containing macropinosomes underwent a full program of maturation, in line with the relatively late timing of VACV MV penetration.…”

Section: Discussionmentioning

confidence: 93%

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Rizopoulos

Balistreri

Kilcher

et al. 2015

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The prototypic poxvirus, vaccinia virus (VACV), occurs in two infectious forms, mature virions (MVs) and extracellular virions (EVs). Both enter HeLa cells by inducing macropinocytic uptake. Using confocal microscopy, live‐cell imaging, targeted RNAi screening and perturbants of endosome maturation, we analyzed the properties and maturation pathway of the macropinocytic vacuoles containing VACV MVs in HeLa cells. The vacuoles first acquired markers of early endosomes [Rab5, early endosome antigen 1 and phosphatidylinositol(3)P]. Prior to release of virus cores into the cytoplasm, they contained markers of late endosomes and lysosomes (Rab7a, lysosome‐associated membrane protein 1 and sorting nexin 3). RNAi screening of endocytic cell factors emphasized the importance of late compartments for VACV infection. Follow‐up perturbation analysis showed that infection required Rab7a and PIKfyve, confirming that VACV is a late‐penetrating virus dependent on macropinosome maturation. VACV EV infection was inhibited by depletion of many of the same factors, indicating that both infectious particle forms share the need for late vacuolar conditions for penetration.

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Section: Discussionmentioning

confidence: 93%

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Rizopoulos

Balistreri

Kilcher

et al. 2015

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“…Since LysoTracker dyes also label other low-pH organelles like synaptic vesicles (55), we further discriminated whether the puncta were lysosomes by surveying the late endosome/lysosome marker Rab7. Rab7 transiently binds to late endosomal and lysosomal membranes (56,57). In transgenic zebrafish that express GFPRab7 under a ubiquitous promoter, GFP-Rab7 is expressed in all cells (not just HCs), and a large fraction of the protein is cytosolic.…”

Section: Resultsmentioning

confidence: 99%

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

Hailey¹,

Esterberg²,

Linbo³

et al. 2016

Journal of Clinical Investigation

105

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“…On short time scales (t < ~5 s), the vesicles are caught in the cytoskeleton either in cages or directly attached to a given filament, resulting in anomalous subdiffusion of vesicles (Figure 6B-D).We used a frame rate of ~1 Hz for our tracking studies, which is comparable or even faster than many other vesicle tracking studies with fluorescent probes [39,47,48], even though some vesicle tracking studies use much higher frame rates up to 30 Hz [49]. In our previous work on tracking of sterol vesicles using DHE on a WF microscope, much lower frame rates of ~0.015-0.03 Hz were used to catch significant vesicle displacements in the presence of the unavoidable photobleaching of that sterol probe [3,10].…”

Section: Discussionmentioning

confidence: 99%

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

et al. 2012

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BackgroundCholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport.ResultsWe found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%.ConclusionsThe mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.

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Endo-Lysosomal Vesicles Positive for Rab7 and LAMP1 Are Terminal Vesicles for the Transport of Dextran

Cited by 107 publications

References 44 publications

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Vaccinia Virus Infection Requires Maturation of Macropinosomes

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

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