RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells

Hertel, Fabian; Mo, Gary; Duwé, Sam; Dedecker, Peter; Zhang, Jin

doi:10.1016/j.celrep.2015.12.036

Cited by 53 publications

(52 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells based on statistical analysis of the fluorescence fluctuations. This strategy, termed reconstituted fluorescence-based SOFI (refSOFI), was used to investigate the interaction between the endoplasmic reticulum (ER) Ca 2+ sensor STIM1 and the pore-forming channel subunit ORAI1 at the ER-plasma membrane junction [87]. Limitations of these BiFC-based approaches include irreversibility and long maturation time ranging from 30 min to hours.…”

Section: Resolving Biochemical Activities In Superresolutionmentioning

confidence: 99%

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

Rodriguez

Campbell

Lin

et al. 2017

Trends in Biochemical Sciences

Self Cite

562

451

View full text Add to dashboard Cite

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we attempt to provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

show abstract

Section: Resolving Biochemical Activities In Superresolutionmentioning

confidence: 99%

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

Rodriguez

Campbell

Lin

et al. 2017

Trends in Biochemical Sciences

Self Cite

562

451

View full text Add to dashboard Cite

show abstract

“…Importantly, these findings are largely consistent with our biochemical analysis. 4 For a more robust subunit (38). However, this approach is unsuited for resolving the stoichiometry of higher order protein assemblies such as those formed by SMSr.…”

Section: Discussionmentioning

confidence: 99%

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Cabukusta¹,

Köhlen²,

Richter

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by George CarmanSingle-molecule photobleaching has emerged as a powerful non-invasive approach to extract the stoichiometry of multimeric membrane proteins in their native cellular environment. However, this method has mainly been used to determine the subunit composition of ion channels and receptors at the plasma membrane. Here, we applied single-molecule photobleaching to analyze the oligomeric state of an endoplasmic reticulum (ER) resident candidate ceramide sensor protein, SMSr/SAMD8. Coimmunoprecipitation and chemical cross-linking studies previously revealed that the N-terminal sterile alpha motif (or SAM) domain of SMSr drives self-assembly of the protein into oligomers and that SMSr oligomerization is promoted by curcumin, a drug known to perturb ER ceramide and calcium homeostasis. Application of cell spreading surface-active coating materials in combination with total internal reflection fluorescence (TIRF) microscopy allowed us to image GFP-tagged SMSr proteins as single fluorescent spots in the ER of HeLa cells in which expression of endogenous SMSr was abolished. In line with our biochemical analysis, we find that the number of bleaching steps in SMSr-GFP-positive spots displays a substantial drop after removal of the SAM domain. In contrast, treatment of cells with curcumin increased the number of bleaching steps. Our results document the first successful application of single-molecule photobleaching to resolve drug-induced and domain-dependent changes in the oligomeric state of an ER-resident membrane protein, hence establishing a complementary method to unravel the mechanism by which SMSr controls ceramide levels in the ER.

show abstract

“…The SOFI framework even allows for multiplexing of several emitters, with highly similar steady-state fluorescence characteristics, using differences in their blinking behavior [18]. In addition to the conventional visualization of fluorophore distributions, SOFI has also been used to visualize biosensor activities with similar spatial resolution enhancements [19,20].…”

Section: Introductionmentioning

confidence: 99%

Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI)

et al. 2019

Self Cite

View full text Add to dashboard Cite

Super-resolution (SR) fluorescence microscopy is typically carried out on research microscopes equipped with high-NA TIRF objectives and powerful laser light sources. Super-resolution optical fluctuation imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing SR imaging techniques. Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFIspecific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

show abstract

RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells

Cited by 53 publications

References 59 publications

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

Monitoring Changes in the Oligomeric State of a Candidate Endoplasmic Reticulum (ER) Ceramide Sensor by Single-molecule Photobleaching

Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI)

Contact Info

Product

Resources

About