Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Lajoie, Patrick; Moir, Robyn D.; Willis, Ian M.; Snapp, Erik L.

doi:10.1091/mbc.e11-12-0995

Cited by 89 publications

(112 citation statements)

References 59 publications

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“…Because csiLSFM is designed to work with fluorescently labeled biological specimens, we recorded images of GFP-tagged endoplasmic reticulum in wild-type yeast (BY4741, transformed with pRS415-ER-sfGFP-HDEL) (30). We directly embedded the live yeast cells in 1.5% low-melt agarose.…”

Section: Resultsmentioning

confidence: 99%

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Chang

Meza

Stelzer

2017

Proc. Natl. Acad. Sci. U.S.A.

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Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFM performs with physiologically relevant specimens.he prime reason to use fluorescence light microscopy is the observation of live, thick, multicellular, and 3D specimens under close to natural conditions. Thus, one has to work with specimens that are mounted in an aqueous medium and not attached to or established close to a coverslip. In addition, the main issues in fluorescence microscopy, fluorophore bleaching, endogenous organic molecule phototoxicity, and solar-level intensities have to be addressed (1).Light-sheet-based fluorescence microscopy (LSFM) has emerged as one of the most valuable novel tools in developmental biology (1-5), plant biology (6), and 3D cell biology (7). In contrast to an epifluorescence arrangement, LSFM uses at least two independently operated microscope objective lenses. The lenses used in the excitation of the fluorophores are arranged at an angle of 90°rela-tive to those used for the detection of the 3D fluorophore density distribution. In addition, special optical arrangements ensure that only a thin planar section centered on the focal planes of the detection lenses receives light. Hence, optical sectioning (8, 9), which is not available in conventional fluorescence microscopy, as well as no phototoxicity and no fluorophore bleaching outside a small volume close to the focal plane, are intrinsic properties of LSFM. The energy required to excite the fluorophores when recording a 3D stack of images can be reduced by two to four orders of magnitude relative to wide-field, confocal, and two-photon fluorescence microscopy (10, 11). Modern cameras are used to record millions of pixels in parallel, i.e., tens to hundreds of images with subcellular resolution within a few seconds.Because the lateral resolution of LSFM is similar to that of a conventional epifluorescence microscope, superresolution techniques have been combined with LSF...

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Section: Resultsmentioning

confidence: 99%

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Chang

Meza

Stelzer

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In yeast, these reporters consist of promoters containing UPR elements (UPRE) fused to FPs such as GFP or mCherry [61] ( Figure 2 ). Alternatively, FPs containing an appropriate ER retrieval motif (HDEL) can be chromosomally inserted to tag UPR targets, such as Kar2 [62, 63]. When Kar2-sfGFP-HDEL is expressed under its endogenous promoter, fluorescence levels will also reflect activation of the UPR.…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

“…Expression of transcriptional reporters can be monitored by fluorescence microscopy and flow cytometry, allowing high content monitoring of UPR activation under various conditions (pharmacological stressors, deletion mutants) [62]. A similar approach can be used in model organisms.…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

“…We have shown that this method can detect early changes in the ER misfolded protein burden that cannot be detected via classical UPR assays [81]. Mobility of the yeast BiP homologue, Kar2 can also report on the ER misfolded protein burden [62] ( Figure 4 ). Interestingly, we have shown that Kar2 mobility can reveal different modes of Ire1 activation.…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

“…Interestingly, we have shown that Kar2 mobility can reveal different modes of Ire1 activation. In yeast, inositol depletion can trigger UPR activation in the absence of significant changes in the ER misfolded protein burden [62]. The ability of membrane lipid perturbations to induce UPR independently of accumulation of misfolded proteins has been described using genetic and biochemical methods in both yeast and mammalian cells [25, 83].…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

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Approaches to imaging unfolded secretory protein stress in living cells

Lajoie

Fazio

Snapp

2014

Endoplasmic Reticulum Stress in Diseases

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The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

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Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

Costantini

Snapp

2013

CP Cell Biology

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This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.

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Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Cited by 89 publications

References 59 publications

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm

Approaches to imaging unfolded secretory protein stress in living cells

Probing Endoplasmic Reticulum Dynamics using Fluorescence Imaging and Photobleaching Techniques

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