Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane.

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding.

Cytosolic GAPDH as a redox-dependent regulator of energy metabolism.

Background: Plant cytosolic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GapC) displays redox-dependent changes in its subcellular localizations and activity. Apart from its fundamental role in glycolysis, it also exhibits moonlighting properties. Since the exceptional redox-sensitivity of GapC has been suggested to play a crucial role in its various functions, we here studied its redox-dependent subcellular localization and the influence of the redox-state on GapC protein interactions.

How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins.

The spatiotemporal organisation of membranes is often characterised by the formation of large protein clusters. In Escherichia coli, outer membrane protein (OMP) clustering leads to OMP islands, the formation of which underpins OMP turnover and drives organisation across the cell envelope. Modelling how OMP islands form in order to understand their origin and outer membrane behaviour has been confounded by the inherent difficulties of simulating large numbers of OMPs over meaningful timescales.

Multi-functional DNA nanostructures that puncture and remodel lipid membranes into hybrid materials.

Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers.

Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments.

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization.

Spatiotemporally Controlled Reorganization of Signaling Complexes in the Plasma Membrane of Living Cells.

Triggered immobilization of proteins in the plasma membrane of living cells into functional micropatterns is established by using an adaptor protein, which is comprised of an antiGFP nanobody fused to the HaloTag protein. Efficient in situ reorganization of the type I interferon receptor subunits as well as intact, fully functional signaling complexes in living cells are achieved by this method.

Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions.

We present an ultrasensitive technique for quantitative protein-protein interaction analysis in a two-dimensional format based on phase-separated, micropatterned membranes. Interactions between proteins captured to lipid probes via an affinity tag trigger partitioning into the liquid-ordered phase, which is readily quantified by fluorescence imaging. Based on a calibration with well-defined low-affinity protein-protein interactions, equilibrium dissociation constants >1 mM were quantified.

Rupture of Stochastically Occurring Vesicle Clusters Limits Bilayer Formation on Alkane-PEG-Type Supports: Uncoupling Clustering from Surface Coverage.

Polymer-supported bilayers (PSBs) are a recognized tool for drug discovery through function-interaction analysis of membrane proteins. While silica-supported bilayers (SSBs) spontaneously form from surface-adsorbed vesicles, successful PSB formation via a similar method has thus far been limited by an insufficient understanding of the underlying vesicle-remodelling processes. Here, we generated a polymer support through the incubation of poly-L-lysine conjugated to alkyl-chain-terminated poly(ethylene)glycol on silica.

Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.

Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat.

Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster.

The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS) of Drosophila, neural stem cells (neuroblasts) exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly.

Composition of a neuromere and its segmental diversification under the control of Hox genes in the embryonic CNS of Drosophila.

Studies performed at the level of single, identified cells in the fruitfly Drosophila have decisively contributed to our understanding of the mechanisms underlying the development and function of the nervous system. This review highlights some of the work based on single-cell analyses in the embryonic/larval CNS that sheds light on the principles underlying formation and organization of an entire segmental unit and its divergence along the anterior/posterior body axis.

Monofunctional stealth nanoparticle for unbiased single molecule tracking inside living cells.

On the basis of a protein cage scaffold, we have systematically explored intracellular application of nanoparticles for single molecule studies and discovered that recognition by the autophagy machinery plays a key role for rapid metabolism in the cytosol. Intracellular stealth nanoparticles were achieved by heavy surface PEGylation. By combination with a generic approach for nanoparticle monofunctionalization, efficient labeling of intracellular proteins with high fidelity was accomplished, allowing unbiased long-term tracking of proteins in the outer mitochondrial membrane.

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 (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo 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 ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA.

Abdominal-B and caudal inhibit the formation of specific neuroblasts in the Drosophila tail region.

The central nervous system of Drosophila melanogaster consists of fused segmental units (neuromeres), each generated by a characteristic number of neural stem cells (neuroblasts). In the embryo, thoracic and anterior abdominal neuromeres are almost equally sized and formed by repetitive sets of neuroblasts, whereas the terminal abdominal neuromeres are generated by significantly smaller populations of progenitor cells. Here we investigated the role of the Hox gene Abdominal-B in shaping the terminal neuromeres.

Neuroblast pattern and identity in the Drosophila tail region and role of doublesex in the survival of sex-specific precursors.

The central nervous system is composed of segmental units (neuromeres), the size and complexity of which evolved in correspondence to their functional requirements. In Drosophila, neuromeres develop from populations of neural stem cells (neuroblasts) that delaminate from the early embryonic neuroectoderm in a stereotyped spatial and temporal pattern. Pattern units closely resemble the ground state and are rather invariant in thoracic (T1-T3) and anterior abdominal (A1-A7) segments of the embryonic ventral nerve cord.

Spatial organization of lipid phases in micropatterned polymer-supported membranes.

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l(d)) and liquid-ordered (l(o)) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet.

Diffusion and interaction dynamics of individual membrane protein complexes confined in micropatterned polymer-supported membranes.

Micropatterned polymer-supported membranes (PSM) are established as a tool for confining the diffusion of transmembrane proteins for single molecule studies. To this end, a photochemical surface modification with hydrophobic tethers on a PEG polymer brush is implemented for capturing of lipid vesicles and subsequent fusion. Formation of contiguous membranes within micropatterns is confirmed by scanning force microscopy, fluorescence recovery after photobleaching (FRAP), and super-resolved single-molecule tracking and localization microscopy.