Versatile protein tagging in cells with split fluorescent protein

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives