Improved deconvolution of very weak confocal signals

Day, Kasey J.; Rivière, Patrick J. La; Chandler, Talon; Bindokas, Vytas P.; Ferrier, Nicola; Glick, Benjamin S.

doi:10.12688/f1000research.11773.1

Cited by 13 publications

(12 citation statements)

References 18 publications

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“…4D confocal movies were collected on a Leica SP5 or SP8 microscope using an 80 nm pixel size and a 0.25-0.30 μm Z-step interval and 20-30 optical sections, with a Z-stack collected every 0.5-2.0 s. Static images were converted to 16-bit and average projected, then range-adjusted to the minimum and maximum pixel values in ImageJ (http://rsbweb.nih.gov/ij/). To process movies, a Gaussian blur with radius 0.75 pixels was applied in ImageJ to fluorescence channels (Day et al, 2016), which were then deconvolved with Huygens Essential (Scientific Volume Imaging, Hilversum, The Netherlands) using the CMLE (Classical Maximum Likelihood Estimation) algorithm (Day et al, 2017), and then corrected for bleaching using an ImageJ plugin (cmci.embl.de/downloads/bleach_corrector). Movies were converted to hyperstacks and average projected, then range-adjusted to maximize contrast in ImageJ.…”

Section: Methods Detailsmentioning

confidence: 99%

Budding Yeast Has a Minimal Endomembrane System

2018

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The endomembrane system consists of the secretory and endocytic pathways, which communicate by transport to and from the trans-Golgi network (TGN). In mammalian cells, the endocytic pathway includes early, late, and recycling endosomes. In budding yeast, different types of endosomes have been described, but the organization of the endocytic pathway has been unclear. We performed a spatial and temporal analysis of yeast endosomal markers and endocytic cargoes. Our results indicate that the yeast TGN also serves as an early and recycling endosome. In addition, as previously described, yeast contains a late or prevacuolar endosome (PVE). Endocytic cargoes localize to the TGN shortly after internalization, and manipulations that perturb export from the TGN can slow the passage of endocytic cargoes to the PVE. Yeast apparently lacks a distinct early endosome. Thus, yeast has a simple endocytic pathway that may reflect the ancestral organization of the endomembrane system. eTOC Blurb Although mammalian endocytic pathway has early, late, and recycling endosomes, the Saccharomyces cerevisiae system is unresolved. Day et al. show that yeast only has two endosome types: the trans-Golgi network, functioning as both early and recycling endosome, and the longlived prevacuolar endosome, possibly reflecting ancestral organization of the endomembrane system.

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Section: Methods Detailsmentioning

confidence: 99%

Budding Yeast Has a Minimal Endomembrane System

2018

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“…Huygens Essential (Scientific Volume Imaging) using the classic maximum likelihood estimation 466 algorithm (Day et al, 2017). Movies were converted to hyperstacks and average projected, then 467 range-adjusted to maximize contrast in ImageJ.…”

Section: Yeast Growth and Strain Construction 431mentioning

confidence: 99%

A microscopy-based kinetic analysis of yeast vacuolar protein sorting

Glick

Casler

2020

Preprint

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The yeast Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell 1 fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic 2 through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during 3Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To 4 address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar 5 targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive 6 during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi 7 near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are 8 cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked 9 biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results 10 imply that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run 11 mechanism. 12

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“…Live-cell dual-color 4D confocal imaging was performed previously described (Day et al, 2017) using a strain expressing GFP-Vig4 as an early Golgi marker and Sec7-DsRed as a late Golgi marker, with the following modifications. Cells attached to the cover glass dish surface were washed and covered with minimal SD medium.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

The golgin PpImh1 mediates reversible cisternal stacking in the Golgi of the budding yeast Pichia pastoris

Jain

Dahara

Bhattacharyya

2019

Journal of Cell Science

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The adhesive force for cisternal stacking of Golgi needs to be reversibleto be initiated and undone in a continuous cycle to keep up with the cisternal maturation. Microscopic evidence in support of such a reversible nature of stacking, in the form of 'TGN peeling,' has been reported in various species, suggesting a potential evolutionarily conserved mechanism. However, knowledge of such mechanism has remained sketchy. Here, we have explored this issue in the budding yeast Pichia pastoris which harbors stacked Golgi. We observed that deletion of GRIP domain golgin P. pastoris (Pp)IMH1 increases the peeling of late cisterna, causing unstacking of the Golgi stack. Our results suggest that the PpImh1 dimer mediates reversible stacking through a continuous association-dissociation cycle of its GRIP domain to the middle and late Golgi cisterna under the GTP hydrolysis-based regulation of Arl3-Arl1 GTPase cascade switch. The reversible cisternal stacking function of PpImh1 is independent of its vesicle-capturing function. Since GRIP domain proteins are conserved in plants, animals and fungi, it is plausible that this reversible mechanism of Golgi stacking is evolutionarily conserved. This article has an associated First Person interview with the first author of the paper.

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Improved deconvolution of very weak confocal signals

Cited by 13 publications

References 18 publications

Budding Yeast Has a Minimal Endomembrane System

Budding Yeast Has a Minimal Endomembrane System

A microscopy-based kinetic analysis of yeast vacuolar protein sorting

The golgin PpImh1 mediates reversible cisternal stacking in the Golgi of the budding yeast Pichia pastoris

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