Ivan R. Corrêa

An Engineered Protein Tag for Multiprotein Labeling in Living Cells

The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

A semi-synthetic organism with an expanded genetic alphabet

Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A–T and G–C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS–dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.

Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP‐tag fusion proteins in living cells. SNAP‐tag is an engineered variant of the human repair protein O6‐alkylguanine‐DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP‐tag protein. We describe the use of intramolecularly quenched probes for wash‐free labeling of cell surface‐localized epidermal growth factor receptor (EGFR) fused to SNAP‐tag and for direct quantification of SNAP‐tagged β‐tubulin in cell lysates. In addition, we have characterized a fast‐labeling variant of SNAP‐tag, termed SNAPf, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAPf and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Two-color STED microscopy in living cells.

Diffraction-unlimited resolution provided by Stimulated Emission Depletion (STED) microscopy allows for imaging cellular processes in living cells that are not visible by conventional microscopy. However, it has so far not been possible to study dynamic nanoscale interactions because multicolor live cell STED microscopy has yet to be demonstrated and suitable labeling technologies and protocols are lacking. Here we report the first realization of two-color STED imaging in living cells. Using improved SNAPf and CLIPf technologies to label epidermal growth factor (EGF) and EGF receptor (EGFR), we report resolutions of 78 nm and 82 nm for 22 sequential two-color scans in living cells.

Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA

Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(ii)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson–Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.

Indo-1 derivatives for local calcium sensing.

The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduce here a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in single cells dissociated from muscle of adult mice expressing a nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by shifted excitation and emission ratioing of confocal microscopic images of fluorescence. After permeabilizing the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.

Clathrin-independent pathways do not contribute significantly to endocytic flux

Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells. DOI: http://dx.doi.org/10.7554/eLife.03970.001

The Formin Daam1 and Fascin Directly Collaborate to Promote Filopodia Formation

Filopodia are slender cellular protrusions that dynamically extend and retract to facilitate directional cell migration, pathogen sensing, and cell-cell adhesion. Each filopodium contains a rigid and organized bundle of parallel actin filaments, which are elongated at filopodial tips by formins and Ena/VASP proteins. However, relatively little is known about how the actin filaments in the filopodial shaft are spatially organized to form a bundle with appropriate dimensions and mechanical properties. Here, we report that the mammalian formin Daam1 (Disheveled-associated activator of morphogenesis 1) is a potent actin-bundling protein and localizes all along the filopodial shaft, which differs from other formins that localize specifically to the tips. Silencing of Daam1 led to severe defects in filopodial number, integrity, and architecture, similar to silencing of the bundling protein fascin. This led us to investigate the potential relationship between Daam1 and fascin. Fascin and Daam1 coimmunoprecipitated from cell extracts, and silencing of fascin led to a striking loss of Daam1 localization to filopodial shafts, but not tips. Furthermore, purified fascin bound directly to Daam1, and multicolor single-molecule TIRF imaging revealed that fascin recruited Daam1 to and stabilized Daam1 on actin bundles in vitro. Our results reveal an unanticipated and direct collaboration between Daam1 and fascin in bundling actin, which is required for proper filopodial formation.

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI: http://dx.doi.org/10.7554/eLife.01008.001