Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

Abstract

Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities. Insight, innovation, integration Fluorescent proteins enable imaging in situ, throughout the visible spectrum, with superb molecular specificity and single-molecule sensitivity. Unfortunately, when compared to leading small-molecule fluorophores (e.g., Cy3), fluorescent proteins, suffer from accelerated photobleaching and poor integrated photon output. This results from a lack of appropriate high-throughput methods for improving the photostability of fluorescent proteins, as well as a poor molecular understanding of fluorescent protein photobleaching. Here, we report the first application of a recently developed microfluidic cell-sorter to identify fluorescent proteins from a mCherry-derived library with improved photostability. The results provide insight into fluorescent protein photophysics, greatly accelerate identification of improved mutants, and can be applied to both genetically encoded and small-molecule fluorophores.

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

“…These features of our cell picker will enable it to be deployed easily into a wide variety of protein engineering contexts, without requiring the custom fabrication involved with other strategies such as microfluidics 5 – 7 or laser-released micropallets 36 . For example, microfluidic devices require custom microfabrication, and can require redesign and reimplementation when screening assays or criteria are changed, which in turn can be challenging for many protein engineers 7 , 37 . Here, by choosing a strategy that essentially automates the manual steps done by a molecular engineer, and by implementing it with off-the-shelf parts that are easy to set up, and easily customized software, we aim to not only enable powerful multidimensional screens but to democratize the process of performing them.…”

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

“…For example, this technique could be extremely useful in the screening of novel complex fluorescent proteins for super-resolution microscopy, including photoactivatable or photoconvertible fluorescent proteins [ 11 , 12 ]; or may even be used to select out for fluorescent proteins with more desirable properties such as protection from photobleaching. Current techniques available for screening fluorescent proteins [ 26 ] tend to use relatively slow, serial interrogation methods and do not therefore scale well to very high throughput unlike the high-speed multiplex deposition and parallel analyses method described here.…”

High-throughput, image-based screening of pooled genetic-variant libraries

“…However, unlike screening of individually constructed variants where the genotype is known a priori , screening of pooled libraries requires methods to measure the genotype that produced the desired phenotype, and this is typically done by selecting/enriching library members with desired phenotypes and then using sequencing to determine their corresponding genotypes. Such approaches have been used to identify protein variants with desired properties, such as fluorescent proteins with improved brightness 3 , reversible photoswitching 4 , 5 and increased lifetime 6 – 8 . At the genome scale, RNAi or CRISPR-based approaches have been used in pooled library screens to measure the role of numerous genes in cellular phenotypes such as viability 9 and, more recently, in the expression of the transcriptome 10 – 12 .…”