Xiaokun Shu

Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome.

Infrared Vision Proteins from jellyfish and corals that fluoresce in the visible wavelength range have revolutionized optical imaging of cells. However, these wavelengths are absorbed by hemoglobin, water, and lipids and the proteins are thus not appropriate for deep-tissue imaging. Now Shu et al. (p. 804) have engineered a bacteriophytochrome from Deinococcus radiodurans that incorporates biliverdin as the chromophore, to fluoresce with excitation and emission spectra of 648 and 708 nanometers, respectively. These infrared fluorescent proteins are expressed well in mammalian cells and mice, and can be used for whole-body imaging. An engineered infrared fluorescent protein derived from an extremophile bacterium gives a strong signal in mammalian cells. A bacteriophytochrome incorporating biliverdin has been engineered to generate strong infrared fluorescence in mammalian cells and whole mice. Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M−1 cm−1; and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.

A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG

We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

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 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.

Enhancing Serial Block-Face Scanning Electron Microscopy to Enable High Resolution 3-D Nanohistology of Cells and Tissues

Serial block face scanning electron microscopy (SBFSEM) is a powerful technique originally introduced by Leighton [1], substantially improved by Denk [2] and subsequently commercialized (Gatan Inc., Pleasanton, CA.). SBFSEM allows for the automated image acquisition of relatively large volumes of tissue at near nanometer-scale resolution, using a dry cutting ultramicrotome fitted into an SEM. In an automated process, a low voltage backscatter electron (BSE) image is obtained from the surface of an epoxy embedded tissue block face. The ultramicrotome then removes an ultra-thin section of tissue with a specially designed oscillating diamond knife (Diatome AG, Switzerland), and a block face image from the corresponding region is again obtained. This sequence is repeated over and over until the desired volume of tissue has been imaged. Although SBFSEM overcomes many obstacles routinely encountered with serial section TEM reconstruction, until recently there was a significant limitation to the resolution obtainable by this method compared to conventional TEM. This was due primarily to difficulties encountered using BSE imaging at low accelerating voltages. To overcome this we have developed a protocol for vastly increasing the heavy metal staining of specimens to improve BSE yield. This is accomplished by combining a variety of preexisting heavy metal staining methodologies not normally used together, including ferrocyanide-reduced osmium tetroxide, thiocarbohydrazide-osmium tetroxide (OTO), prolonged uranyl acetate treatment and en bloc lead aspartate staining. Using this approach, we demonstrate a dramatic improvement in image contrast and resolution from existing methods in a variety of specimens (Fig. 1).

An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.

An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V.

Wild‐type green fluorescent protein (wt‐GFP) has a prominent absorbance band centered at ∼395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited‐state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen‐bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time‐resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place ∼30 times more slowly than in wt‐GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen‐bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine‐based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt‐GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation–emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.