Measuring Protein Mobility by Photobleaching <scp>GFP</scp> Chimeras in Living Cells

Snapp, Erik L.; Altan, Nihal; Lippincott‐Schwartz, Jennifer

doi:10.1002/0471143030.cb2101s19

Cited by 108 publications

(134 citation statements)

References 37 publications

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“…No photobleaching of the cell or adjacent cells during fluorescence recovery was observed. Diffusion (D) measurements were calculated as described previously (Siggia et al, 2000;Snapp et al, 2003a). Composite figures were prepared using Photoshop CS2 and Illustrator CS software (Adobe).…”

Section: Fluorescence Recovery After Photobleaching Analysesmentioning

confidence: 99%

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Heyden

Naismith

Snapp

et al. 2009

MBoC

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115

162

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TorsinA (TorA) is an AAA؉ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia.

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Section: Fluorescence Recovery After Photobleaching Analysesmentioning

confidence: 99%

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Heyden

Naismith

Snapp

et al. 2009

MBoC

Self Cite

115

162

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“…A nucleoplasmic ROI (4 Â 4 mm) was defined, and 20 pre-bleach 256 Â 256 pixel images were acquired followed by ten ROI bleach cycles (frames captured while zoomed in on ROI and using 100% of 458-, 476-and 488-nm laser line power) over 3.8 s. Recovery was followed over 100 frames acquired at 2.6 frames/s, and then over a further 100 frames acquired every 2 s when recovery was slow. Protein mobility (t 1/2 ) was calculated as reported previously [19,35]. …”

mentioning

confidence: 99%

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

2009

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Sterile inflammation is a host response to tissue injury that is mediated by damage-associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro-inflammatory cytokine IL-1a is reported to be a damage-associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL-1a is produced by microglia, the resident brain macrophages. We found that IL-1a is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL-1a to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygenglucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL-1a released significantly less IL-1a than microglia with predominantly cytosolic IL-1a. The remaining IL-1a was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL-1a may serve to limit inflammation following cell death.Key words: IL-1a . Microglia . Necrosis . Nuclear retention . Sterile inflammation Supporting Information available online IntroductionSterile inflammation is a host response to tissue injury that can exacerbate the initial insult [1]. Intracellular molecules released from necrotic cells act as damage-associated molecular patterns (DAMP), signaling to the innate immune system that tissue injury has occurred [2]. IL-1a, a pro-inflammatory member of the IL-1 cytokine family [3], has recently been identified as a DAMP released from necrotic cells [4]. In addition, necrotic cell DAMP are reported to stimulate IL-1a production by immune cells, which further exacerbates sterile inflammation [1]. Experimental stroke in rodents elicits an inflammatory response that markedly exacerbates brain injury [5]. IL-1a is a key contributor to this injury, and its genetic ablation (along with IL-1b, a related cytokine) causes a 70% reduction in the brain injury caused by stroke [6].In the brain after injury (e.g. stroke, hemorrhage, trauma), IL-1 family cytokines are produced by microglia, the resident brain macrophages [7]. IL-1a is produced in the cytosol as a 31-kDa protein that, following release from necrotic cells, activates the type I IL-1 receptor on responsive cell membranes [8,9]. The Nterminal domain of IL-1a contains a nuclear localization sequence, which mediates active nuclear import [10,11]. IL-1a has been reported to act within the nucleus to regulate cytokine transcription and RNA splicing [12,13]. In this study we sought to test the hypothesis that IL-1a nuclear import also inhibits IL-1a release following necrotic cell death. We report that IL-1a nuclear import and intranuclear retention reduce IL-1a release from necrotic microglia. Thus, IL-1a nuclear retention by necrotic cells may inhibit injury-induced inflammation. Results IL-1a nuclear localization is inhibited at high cell densityTo investigate the effects of IL-1a nuclear localization on IL-1a release after ne...

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“…Several popular FPs, including EGFP, have a propensity to oligomerize [49, 50] and proteins advertised as “monomeric,” may still oligomerize in cells [47]. This is especially true when the FPs are attached to integral membrane proteins, which confines the FPs to a two-dimensional plane and increases the effective concentration leading to a higher probability of oligomerizing.…”

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

confidence: 99%

“…This is especially true when the FPs are attached to integral membrane proteins, which confines the FPs to a two-dimensional plane and increases the effective concentration leading to a higher probability of oligomerizing. Monomerizing mutations and variants have been reported and these should be used in the design of any fusion proteins [49, 50]. …”

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

confidence: 99%

“…Thus, an increase in molecular size (complex formation) or environmental crowding decreases D . Conversely increased D indicates release of a protein from a complex or decreased viscosity [49]. 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].…”

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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Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells

Cited by 108 publications

References 37 publications

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

Approaches to imaging unfolded secretory protein stress in living cells

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