Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System

We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either reversibly controlling the fluorogen concentration or by irreversibly photobleaching the FAST-fluorogen complex. The experimentally determined resolution of 28 nm allowed us to resolve Ensconsin-labeled microtubules and to track single molecules in mitochondria. Our results demonstrate that FAST is well-suited for single-molecule localization microscopy (SMLM). The small size and the availability of spectrally distinct fluorogens present unique advantages of the FAST system as a potential orthogonal labeling strategy that could be applied in conjunction with existing super-resolution dyes and photoactivatable proteins in versatile imaging applications

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

DataSheet1_A fluorescent reporter system for anaerobic thermophiles.DOCX

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli—Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes.</p

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

Light-Activated Kinases Enable Temporal Dissection of Signaling Networks in Living Cells

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway

CuAAC Functionalization of Azide-Tagged Copper(I)-NHCs Acting as Catalyst and Substrate

Azide-tagged copper­(I) complexes of N-heterocyclic carbenes (NHCs) analogous to the well-known bis­[2,6-diisopropylphenyl]­imidazol-2-ylidene (IPr) and bis­[2,6-diisopropylphenyl]­imidazolin-2-ylidene (SIPr) were synthesized and characterized. These complexes were able to act as catalyst and substrate in a model copper-catalyzed azide–alkyne (CuAAC) reaction with propargyl alcohol, yielding functionalized copper­(I)-NHC complexes

Simplified Preparation of Copper(I) NHCs Using Aqueous Ammonia

The use of aqueous ammonia as a coordinating and basic reagent for the metalation of imidazol­(in)­ium salts derived from (S)­IPr and (S)­IMes with simple inorganic copper precursors is reported. The synthesis of copper carbene complexes was performed using aqueous/alcoholic media in mild conditions (room temperature to 60 °C) with short reaction times. Heteroleptic bromide and iodide complexes could be prepared efficiently for (S)­IPr, while chloride complexes were easily accessible for SIMes and (S)­IPr. Ammonia also allowed the efficient synthesis of homoleptic [Cu­(NHC)₂]­(Y) (NHC = (S)­IMes; Y = PF₆, BF₄) species by a variant procedure in ethanol