Photon‐Induced Near‐Field Electron Microscopy of Eukaryotic Cells

Photon‐induced near‐field electron microscopy (PINEM) is a technique to produce and then image evanescent electromagnetic fields on the surfaces of nanostructures. Most previous applications of PINEM have imaged surface plasmon‐polariton waves on conducting nanomaterials. Here, the application of PINEM on whole human cancer cells and membrane vesicles isolated from them is reported. We show that photons induce time‐, orientation‐, and polarization‐dependent evanescent fields on the surfaces of A431 cancer cells and isolated membrane vesicles. Furthermore, the addition of a ligand to the major surface receptor on these cells and vesicles (epidermal growth factor receptor, EGFR) reduces the intensity of these fields in both preparations. We propose that in the absence of plasmon waves in biological samples, these evanescent fields reflect the changes in EGFR kinase domain polarization upon ligand binding.     

Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for a range of biological studies. Bond‐forming reactions for site‐selective protein labeling are commonly used in these endeavors. Selective bond‐cleavage reactions, however, are much less explored and still pose a great challenge. In addition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells. Reported here is an approach for studying uniquely modified proteins containing a traceless cell delivery unit and palladium‐based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells. This approach is demonstrated for the synthesis of a caged ubiquitin‐aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.     

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris‐NTA) head groups were developed for the selective targeting of His‐tagged proteins into liquid ordered (l_o) or liquid disordered (l_d) lipid phases. Strong partitioning into the l_o phase of His‐tagged proteins bound to tris‐NTA conjugated to saturated alkyl chains (tris‐NTA DODA) was achieved, while tris‐NTA conjugated to an unsaturated alkyl chain (tris‐NTA SOA) predominantly resided in the l_d phase. Interestingly, His‐tag‐mediated lipid crosslinking turned out to be required for efficient targeting into the l_o phase by tris‐NTA DODA. Robust partitioning into l_o phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into l_o and l_d domains within the plasma membrane of living cells was demonstrated by single‐molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.     

High‐Rate,Tunable Syngas Production with Artificial Photosynthetic Cells

An artificial photosynthetic (APS) system consisting of a photoanodic semiconductor that harvests solar photons to split H₂O, a Ni‐SNG cathodic catalyst for the dark reaction of CO₂ reduction in a CO₂‐saturated NaHCO₃ solution, and a proton‐conducting membrane enabled syngas production from CO₂ and H₂O with solar‐to‐syngas energy‐conversion efficiency of up to 13.6 %. The syngas CO/H₂ ratio was tunable between 1:2 and 5:1. Integration of the APS system with photovoltaic cells led to an impressive overall quantum efficiency of 6.29 % for syngas production. The largest turnover frequency of 529.5 h^?1 was recorded with a photoanodic N‐TiO₂ nanorod array for highly stable CO production. The CO‐evolution rate reached a maximum of 154.9 mmol g^?1 h^?1 in the dark compartment of the APS cell. Scanning electrochemical–atomic force microscopy showed the localization of electrons on the single‐nickel‐atom sites of the Ni‐SNG catalyst, thus confirming that the multielectron reduction of CO₂ to CO was kinetically favored.     

High‐Resolution NMR Determination of the Dynamic Structure of Membrane Proteins

¹⁵N spin‐relaxation rates are demonstrated to provide critical information about the long‐range structure and internal motions of membrane proteins. Combined with an improved calculation method, the relaxation‐rate‐derived structure of the 283‐residue human voltage‐dependent anion channel revealed an anisotropically shaped barrel with a rigidly attached N‐terminal helix. Our study thus establishes an NMR spectroscopic approach to determine the structure and dynamics of mammalian membrane proteins at high accuracy and resolution.     

HKOH‐1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells

The hydroxyl radical (^.OH), one of the most reactive and deleterious reactive oxygen species (ROS), has been suggested to play an essential role in many physiological and pathological scenarios. However, a reliable and robust method to detect endogenous ^.OH is currently lacking owing to its extremely high reactivity and short lifetime. Herein we report a fluorescent probe HKOH‐1 with superior in vitro selectivity and sensitivity towards ^.OH. With this probe, we have calibrated and quantified the scavenging capacities of a wide range of reported ^.OH scavengers. Furthermore, HKOH‐1r, which was designed for better cellular uptake and retention, has performed robustly in detection of endogenous ^.OH generation by both confocal imaging and flow cytometry. Furthermore, this probe has been applied to monitor ^.OH generation in HeLa cells in response to UV light irradiation. Therefore, HKOH‐1 could be used for elucidating ^.OH related biological functions.     

Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

A multimodal activity‐based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin‐reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation‐induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle‐targeting of activity‐based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Super‐Chelators for Advanced Protein Labeling in Living Cells

Live‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His₆‐ and His₁₂‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.