Rita Strack

A rapidly maturing far-red derivative of DsRed-Express2 for whole-cell labeling.

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy.

A Noncytotoxic Dsred Variant for Whole-Cell Labeling.

A common application of fluorescent proteins is to label whole cells, but many RFPs are cytotoxic when used with standard high-level expression systems. We engineered a rapidly maturing tetrameric fluorescent protein called DsRed-Express2 that has minimal cytotoxicity. DsRed-Express2 exhibits strong and stable expression in bacterial and mammalian cells, and it outperforms other available RFPs with regard to photostability and phototoxicity.

Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach

Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI), allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Here we characterize the structural features that are required for fluorophore binding to Spinach2 and describe novel fluorophores that bind and are switched to a fluorescent state by Spinach2. These diverse Spinach2–fluorophore complexes exhibit fluorescence that is more compatible with existing microscopy filter sets and allows Spinach2-tagged constructs to be imaged with either GFP or YFP filter cubes. Thus, these “plug-and-play” fluorophores allow the spectral properties of Spinach2 to be altered on the basis of the specific spectral needs of the experiment.

A noncytotoxic DsRed variant for whole-cell labeling

Fluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells and having desirable photophysical properties. Our goal was to create a variant of DsRed-Express that maintains the brightness, fast-maturation, and photostability of this protein, while exhibiting decreased cytotoxicity. For this purpose, we mutated the surface of DsRed-Express to decrease aggregation and created DsRed-Express2. DsRed-Express2 retains the favorable photophysical properties of DsRed-Express while showing dramatically reduced cytotoxicity and higher expression in bacterial and mammalian systems. Further, it was shown that DsRed-Express2 outperforms other red FPs as a label for bacterial and mammalian cells.

Imaging bacterial protein expression using genetically encoded RNA sensors

The difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome by using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins and exhibit fluorescence increases that enable protein expression to be imaged in living Escherichia coli. These sensors are key components of a generalizable strategy to image protein expression in a single bacterium in real time.

Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria

Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.

Chromophore formation in DsRed occurs by a branched pathway.

Like GFP, the fluorescent protein DsRed has a chromophore that forms autocatalytically within the folded protein, but the mechanism of DsRed chromophore formation has been unclear. It was proposed that an initial oxidation generates a green chromophore, and that a final oxidation yields the red chromophore. However, this model does not adequately explain why a mature DsRed sample contains a mixture of green and red chromophores. We present evidence that the maturation pathway for DsRed branches upstream of chromophore formation. After an initial oxidation step, a final oxidation to form the acylimine of the red chromophore is in kinetic competition with a dehydration to form the green chromophore. This scheme explains why green and red chromophores are alternative end points of the maturation pathway.

The Yeast GRASP Grh1 Colocalizes with COPII and Is Dispensable for Organizing the Secretory Pathway

In mammalian cells, the ‘Golgi reassembly and stacking protein’ (GRASP) family has been implicated in Golgi stacking, but the broader functions of GRASP proteins are still unclear. The yeast Saccharomyces cerevisiae contains a single non‐essential GRASP homolog called Grh1. However, Golgi cisternae in S. cerevisiae are not organized into stacks, so a possible structural role for Grh1 has been difficult to test. Here, we examined the localization and function of Grh1 in S. cerevisiae and in the related yeast Pichia pastoris, which has stacked Golgi cisternae. In agreement with earlier studies indicating that Grh1 interacts with coat protein II (COPII) vesicle coat proteins, we find that Grh1 colocalizes with COPII at transitional endoplasmic reticulum (tER) sites in both yeasts. Deletion of P. pastoris Grh1 had no obvious effect on the structure of tER–Golgi units. To test the role of S. cerevisiae Grh1, we exploited the observation that inhibiting ER export in S. cerevisiae generates enlarged tER sites that are often associated with the cis Golgi. This tER–Golgi association was preserved in the absence of Grh1. The combined data suggest that Grh1 acts early in the secretory pathway, but is dispensable for the organization of secretory compartments.

New approaches for sensing metabolites and proteins in live cells using RNA.

Tools to study the abundance, distribution, and flux of intracellular molecules are crucial for understanding cellular signaling and physiology. Although powerful, the current FRET-based technology for imaging cellular metabolites is not easily generalizable. Thus, new platforms for generating genetically encoded sensors are needed. We recently developed a new class of biosensors on the basis of Spinach, an RNA mimic of GFP. In this case, RNA aptamers against a target ligand are modularly fused to Spinach that substantially induce Spinach fluorescence in the presence of ligand. We have used this approach to detect metabolites and proteins both in vitro and in living bacteria, thus providing an alternative to FRET-based sensors and a generalizable approach for generating fluorescent sensors to any ligand of interest.

Highly multiplexed imaging

and high computational costs are being tackled. Researchers in academic settings as well as in startup companies such as Deep Genomics, launched July 22, 2015, by some of the authors of DeepBind, will increasingly apply deep learning to genome analysis and precision medicine. The goal is to predict the effect of genetic variants— both naturally occurring and introduced by genome editing—on a cell’s regulatory landscape and how this in turn affects disease development. Nicole Rusk ❯❯Deep learning