BackgroundHuntington's disease (HD) is a fatal inherited neurodegenerative disease, caused by a

We report on the rational engineering of the binding interface of the self-ligating HaloTag protein to generate an optimized linker for DNA nanostructures. Five amino acids positioned around the active-site entry channel for the chlorohexyl ligand (CH) of the HaloTag protein were exchanged for positively charged lysine amino acids to produce the HOB (halo-based oligonucleotide binder) protein. HOB was genetically fused with the enzyme cytochrome P450 BM3, as well as with BMR, the separated reductase domain of BM3. The resulting HOB-fusion proteins revealed significantly improved rates in ligation with CH-modified oligonucleotides and DNA origami nanostructures. These results suggest that the efficient self-assembly of protein-decorated DNA structures can be greatly improved by fine-tuning of the electrostatic interactions between proteins and the negatively charged nucleic acid nanostructures.

Edited by Wolfgang Peti Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22 phox) and three cytosolic subunits (p40 phox , p47 phox , and p67 phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy basedonFRET-fluorescencelifetimeimaging(FLIM)andfluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter-and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Cyan fluorescent proteins (CFPs) derived from Aequorea victoria green fluorescent protein are the most widely used Förster resonant energy transfer (FRET) donors in genetically encoded biosensors for live-cell imaging and bioassays. However, the weak and complex fluorescence emission of cyan variants, such as enhanced cyan fluorescent protein (ECFP) or Cerulean, has long remained a major bottleneck in these FRET techniques. Recently, several CFPs with greatly improved performances, including mTurquoise, mTurquoise2, mCerulean3, and Aquamarine, have been engineered through a mixture of site-directed and large-scale random mutagenesis. This review summarizes the engineering and relative merits of these new cyan donors, which can readily replace popular CFPs in FRET imaging protocols, while reaching fluorescence quantum yields close to 90%, and unprecedented long, near-single fluorescence lifetimes of about 4 ns. These variants display an increased general photostability and much reduced environmental sensitivity, notably towards acid pH. These new, bright, and robust CFPs now open up exciting outlooks for fluorescence lifetime imaging microscopy and advanced quantitative FRET analyses in living cells. In addition, the stepwise engineering of Aquamarine shows that only two critical mutations in ECFP, and one in Cerulean, are required to achieve these performances, which brings new insights into the structural bases of their photophysical properties.

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. During in vitro differentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation of Lbx1 at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

We present a novel experimental setup, which introduces a solid-immersion-lens (SIL) into a confocal microphotoluminescence system. This non-destructive method allows us, within a field of view of 35 µm, to reach a spatial resolution of about 200 nm, which is comparable to near-field systems. We demonstrate an enhancement of the collection efficiency by a factor of five in comparison to standard confocal setups. This is very important for single-dot spectroscopy, where the excitation and detection signals are very low. Moreover, we prove that the spatial resolution and collection efficiency of the system are quite insensitive to air gaps which could be formed at the interface between sample and SIL. These two features can be explained with theoretical considerations. For all these reasons, the system is very suitable for the study of single excitonic lines in quantum dots. We show its application in polarization-and temperaturedependent studies.

Background and Objectives: Pluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the 'fourth' germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands' development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations. Methods: Murine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level. Results: Comparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved. Conclusions: Our findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

The back cover picture shows “HOB Art”, an artistic depiction of a DNA origami nanostructure decorated with six HOB proteins. HOB is a halo‐based oligonucleotide‐binder protein with a rationally engineered binding interface to attain extraordinarily high reaction rates in the covalent ligation with chlorohexyl‐modified DNA. More information about the art of HOBbing proteins with DNA can be found in the communication by C. M. Niemeyer et al. on page 1102 in Issue 12, 2016 (DOI: 10.1002/cbic.201600039).