A Photoswitchable Ligand Targeting the ß1 -Adrenoceptor Enables Light-Control of the Cardiac Rhythm.

Catecholamine-triggered ß-adrenoceptor (ß-AR) signaling is essential for the correct functioning of the heart. Although both ß1 - and ß2 -AR subtypes are expressed in cardiomyocytes, drugs selectively targeting ß1 -AR have proven this receptor as the main target for the therapeutic effects of beta blockers in the heart. Here, we report a new strategy for the light-control of ß1 -AR activation by means of photoswitchable drugs with a high level of ß1 -/ß2 -AR selectivity. All reported molecules allow for an efficient real-time optical control of receptor function in vitro. Moreover, using confocal microscopy we demonstrate that the binding of our best hit, pAzo-2, can be reversibly photocontrolled. Strikingly, pAzo-2 also enables a dynamic cardiac rhythm management on living zebrafish larvae using light, thus highlighting the therapeutic and research potential of the developed photoswitches. Overall, this work provides the first proof of precise control of the therapeutic target ß1 -AR in native environments using light.

Ultrabright Fluorescent Organic Nanoparticles Based on Small-Molecule Ionic Isolation Lattices*.

Ultrabright fluorescent nanoparticles (NPs) hold great promise for demanding bioimaging applications. Recently, extremely bright molecular crystals of cationic fluorophores were obtained by hierarchical coassembly with cyanostar anion-receptor complexes. These small-molecule ionic isolation lattices (SMILES) ensure spatial and electronic isolation to prohibit aggregation quenching of dyes. We report a simple, one-step supramolecular approach to formulate SMILES materials into NPs. Rhodamine-based SMILES NPs stabilized by glycol amphiphiles show high fluorescence quantum yield (30 %) and brightness per volume (5000 M-1 cm-1 /nm3 ) with 400 dye molecules packed into 16-nm particles, corresponding to a particle absorption coefficient of 4×107  M-1 cm-1 . UV excitation of the cyanostar component leads to higher brightness (>6000 M-1 cm-1 / nm3 ) by energy transfer to rhodamine emitters. Coated NPs stain cells and are thus promising for bioimaging.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells.

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the ß1 adrenergic receptor and the ß2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

Click Chemistry Mediated Functionalization of Vertical Nanowires for Biological Applications.

Semiconductor nanowires (NWs) are gaining significant importance in various biological applications, such as biosensing and drug delivery. Efficient and controlled immobilization of biomolecules on the NW surface is crucial for many of these applications. Here, we present for the first time the use of the Cu(I) -catalyzed alkyne-azide cycloaddition and its strain-promoted variant for the covalent functionalization of vertical NWs with peptides and proteins. The potential of the approach was demonstrated in two complementary applications of measuring enzyme activity and protein binding, which is of general interest for biological studies. The attachment of a peptide substrate provided NW arrays for the detection of protease activity. In addition, green fluorescent protein was immobilized in a site-specific manner and recognized by antibody binding to demonstrate the proof-of-concept for the use of covalently modified NWs for diagnostic purposes using minute amounts of material.

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins.

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8-35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting 'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.

Modulation of fluorescence signals from biomolecules along nanowires due to interaction of light with oriented nanostructures.

High aspect ratio nanostructures have gained increasing interest as highly sensitive platforms for biosensing. Here, well-defined biofunctionalized vertical indium arsenide nanowires are used to map the interaction of light with nanowires depending on their orientation and the excitation wavelength. We show how nanowires act as antennas modifying the light distribution and the emitted fluorescence. This work highlights an important optical phenomenon in quantitative fluorescence studies and constitutes an important step for future studies using such nanostructures.

Covalent and stable CuAAC modification of silicon surfaces for control of cell adhesion.

Stable primary functionalization of metal surfaces plays a significant role in reliable secondary attachment of complex functional molecules used for the interfacing of metal objects and nanomaterials with biological systems. In principle, this can be achieved through chemical reactions either in the vapor or liquid phase. In this work, we compared these two methods for oxidized silicon surfaces and thoroughly characterized the functionalization steps by tagging and fluorescence imaging. We demonstrate that the vapor-phase functionalization only provided transient surface modification that was lost on extensive washing. For stable surface modification, a liquid-phase method was developed. In this method, silicon wafers were decorated with azides, either by silanization with (3-azidopropyl)triethoxysilane or by conversion of the amine groups of an aminopropylated surface by means of the azido-transfer reaction. Subsequently, D-amino acid adhesion peptides could be immobilized on the surface by use of Cu(I)-catalyzed click chemistry. This enabled the study of cell adhesion to the metal surface. In contrast to unmodified surfaces, the peptide-modified surfaces were able to maintain cell adhesion during significant flow velocities in a microflow reactor.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

Active plasma membrane P-type H+-ATPase reconstituted into nanodiscs is a monomer.

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Functionalized amphipols: a versatile toolbox suitable for applications of membrane proteins in synthetic biology.

Amphipols are amphipathic polymers that stabilize membrane proteins isolated from their native membrane. They have been functionalized with various chemical groups in the past years for protein labeling and protein immobilization. This large toolbox of functionalized amphipols combined with their interesting physico-chemical properties give opportunities to selectively add multiple functionalities to membrane proteins and to tune them according to the needs. This unique combination of properties makes them one of the most versatile strategies available today for exploiting membrane proteins onto surfaces for various applications in synthetic biology. This review summarizes the properties of functionalized amphipols suitable for synthetic biology approaches.

Exploring arrays of vertical one-dimensional nanostructures for cellular investigations.

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.

A transparent nanowire-based cell impalement device suitable for detailed cell-nanowire interaction studies.

A method to fabricate inexpensive and transparent nanowire impalement devices is invented based on CuO nanowire arrays grown by thermal oxidation. By employing a novel process the nanowires are transferred to a transparent, cell-compatible epoxy membrane. Cargo delivery and detailed cell-nanowire interaction studies are performed, revealing that the cell plasma membrane tightly wraps the nanowires, while cell membrane penetration is not observed. The presented device offers an efficient investigation platform for further optimization, leading towards a simple and versatile impalement delivery system.

Effects of buffer composition and dilution on nanowire field-effect biosensors.

Nanowire-based field-effect transistors (FETs) can be used as ultra-sensitive and label-free biosensors for detecting protein-protein interactions. A way to increase the performance of such sensors is to dilute the sensing buffer drastically. However, we show here that this can have an important effect on the function of the proteins. Moreover, it is demonstrated that this dilution significantly affects the pH stability of the sensing buffer, which consequently impacts the charge of the protein and thus the response and signal-to-noise ratio in the sensing experiments. Three model systems are investigated experimentally to illustrate the impact on ligand-protein and protein-protein interactions. Simulations are performed to illustrate the effect on the performance of the sensors. Combining various parameters, the current study provides a means for evaluating and selecting the most appropriate buffer composition for bioFET measurements.

Realizing integration in structural biology: The 2022 ISBUC Annual Meeting.

This meeting report presents the 2022 Annual Meeting of the cluster for Integrative Structural Biology at the University of Copenhagen (ISBUC) and discusses the cluster approach to interdisciplinary research management. This approach successfully facilitates cross-faculty and inter-departmental collaboration. Innovative integrative research collaborations ignited by ISBUC, as well as research presented at the meeting, are showcased.

Cell membrane conformation at vertical nanowire array interface revealed by fluorescence imaging.

The perspectives offered by vertical arrays of nanowires for biosensing applications in living cells depend on the access of individual nanowires to the cell interior. Recent results on electrical access and molecular delivery suggest that direct access is not always obtained. Here, we present a generic approach to directly visualize the membrane conformation of living cells interfaced with nanowire arrays, with single nanowire resolution. The method combines confocal z-stack imaging with an optimized cell membrane labelling strategy which was applied to HEK293 cells interfaced with 2-11 µm long and 3-7 µm spaced nanowires with various surface coatings (bare, aminosilane-coated or polyethyleneimine-coated indium arsenide). We demonstrate that, for all commonly used nanowire lengths, spacings and surface coatings, nanowires generally remain enclosed in a membrane compartment, and are thereby not in direct contact with the cell interior.

The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports.

Because of the importance of their physiological functions, cell membranes represent critical targets in biological research. Membrane proteins, which make up approximately 1/3 of the proteome, interact with a wide range of small ligands and macromolecular partners as well as with foreign molecules such as synthetic drugs, antibodies, toxins, or surface recognition proteins of pathogenic organisms. Whether it is for the sake of basic biomedical or pharmacological research, it is of great interest to develop tools facilitating the study of these interactions. Surface-based in vitro assays are appealing because they require minimum quantities of reagents, and they are suitable for multiplexing and high-throughput screening. We introduce here a general method for immobilizing functional, unmodified integral membrane proteins onto solid supports, thanks to amphipathic polymers called "amphipols." The key point of this approach is that functionalized amphipols can be used as universal adapters to associate any membrane protein to virtually any kind of support while stabilizing its native state. The generality and versatility of this strategy is demonstrated by using 5 different target proteins, 2 types of supports (chips and beads), 2 types of ligands (antibodies and a snake toxin), and 2 detection methods (surface plasmon resonance and fluorescence microscopy).

Intact mammalian cell function on semiconductor nanowire arrays: new perspectives for cell-based biosensing.

Nanowires (NWs) are attracting more and more interest due to their potential cellular applications, such as delivery of compounds or sensing platforms. Arrays of vertical indium-arsenide (InAs) NWs are interfaced with human embryonic kidney cells and rat embryonic dorsal root ganglion neurons. A selection of critical cell functions and pathways are shown not to be impaired, including cell adhesion, membrane integrity, intracellular enzyme activity, DNA uptake, cytosolic and membrane protein expression, and the neuronal maturation pathway. The results demonstrate the low invasiveness of InAs NW arrays, which, combined with the unique physical properties of InAs, open up their potential for cellular investigations.

Specific and reversible immobilization of histidine-tagged proteins on functionalized silicon nanowires.

Silicon nanowire (Si NW)-based field effect transistors (FETs) have shown great potential as biosensors (bioFETs) for ultra-sensitive and label-free detection of biomolecular interactions. Their sensitivity depends not only on the device properties, but also on the function of the biological recognition motif attached to the Si NWs. In this study, we show that SiNWs can be chemically functionalized with Ni:NTA motifs, suitable for the specific immobilization of proteins via a short polyhistidine tag (His-tag) at close proximity to the SiNW surface. We demonstrate that the proteins preserve their function upon immobilization onto SiNWs. Importantly, the protein immobilization on the Si NWs is shown to be reversible after addition of EDTA or imidazole, thus allowing the regeneration of the bioFET when needed, such as in the case of proteins having a limited lifetime. We anticipate that our methodology may find a generic use for the development of bioFETs exploiting functional protein assays because of its high compatibility to various types of NWs and proteins.

Intrinsic anti-Stokes emission in living HeLa cells.

Intrinsic fluorescence of biological material, also called auto-fluorescence, is a well-known phenomenon and has in recent years been used for imaging, diagnostics and cell viability studies. Here we show that in addition to commonly observed auto-fluorescence, intrinsic anti-Stokes emission can also be observed under 560 nm or 633 nm excitation. The anti-Stokes emission is shown to be spatially located on/in the mitochondria. The findings presented here show that sensitive imaging experiments e.g. single molecule experiments or two-photon excitation imaging can be compromised if intracellular anti-Stokes emission is not accounted for. On the other hand, we suggest that this anti-Stokes emission could be exploited as an additional modality for mitochondria visualization and cell viability investigation even in systems that are already labeled with commonly used fluorophores that rely on normal Stokes-based detection.

Chemically specific laser-induced patterning of alkanethiol SAMs: characterization by SEM and AFM.

Self-assembled monolayers (SAMs) are widely used to modify the interfacial properties of solid surfaces in either a homogeneous or a patterned manner. One of the many techniques developed for patterning SAMs involves heating the surface with a focused laser beam. Localized heating can result in pattern formation through either the ablation of both the solid substrate and the SAM (chemically nonspecific patterning) or, at lower temperatures, the selective breaking of the chemical bonds between the SAM and the substrate (chemically specific patterning). The latter method is termed chemically specific laser-induced patterning and is demonstrated for alkanethiol monolayers on gold (Au). In this report, the interplay between alkanethiol desorption and nanoscale Au ablation is studied using atomic force microscopy to image both the topographical and the chemical features of laser patterned areas. Frequently the two processes occur simultaneously but with different spatial extents, as predicted theoretically, due to their different threshold temperatures. By tuning the exposure conditions (laser power and irradiation time), parameters are established where local heating causes alkanethiol desorption without any Au ablation, thus, allowing chemically specific desorption and patterning of alkanethiol SAMs. This allows chemical patterns to be created without changes in the surface topography. Using scanning electron microscopy, a linear dependence of pattern size on irradiation time is demonstrated for circular features 0.5-1 microm in diameter.