Research Programme
Rationally designed surface architectures for nanoscale interrogation and manipulation of biomolecules at membranes
The research idea of this RTG is to develop novel quantitative biophysical techniques to resolve dynamic processes at membranes from mesoscopic down to atomic scales. The starting point are emerging nanomaterials with tuneable electronic and optical properties, which have recently found striking applications in biology. With their capability of energy confinement in nanoscale dimensions, these materials offer a huge potential for non-invasive manipulation of biological processes with unprecedented spatial and temporal resolution. To exploit this potential, the RTG will establish a network of collaborative doctoral projects integrating the comprehensive expertise in nanomaterial synthesis, functional characterization as well as bio-functionalization and application in membrane biology.
Project Cluster I
Energy-converting nanoparticles for interrogating lipid dynamics at signalling complexes in the plasma membrane
The overall objective of PC-I is to exploit the unique capa-bility of upconversion nanoparticles (UCNP) to highly se-lectively excite chromophores only in very close proximity of target proteins via lanthanide resonance energy trans-fer (ucLRET). For this purpose, we will engineer UCNP with different sizes and particle architectures to ensure maximum LRET efficiency (Project 1). In conjunction with optimized excitation schemes using the capabilities of ultrafast laser pulsing (Project 2) ucLRET efficiencies for detection at single molecule level will be achieved. By integrating the UCPN into surface architectures that enable capturing of signalling complexes in live cells (Project 3), we will interface these with signalling complexes in the plasma membrane, which will be captured via extracellular affinity tags. The dynam-ics of fluorescent lipid probes in the ultimate vicinity of the captured proteins will be probed by fluorescence correlation spectroscopy (FCS) using ucLRET for nanoscale confinement of fluo-rescence excitation (ucFCS).
Project Cluster II
Supramolecular surface architectures for directing and interrogating plasma membrane permeabilization and repair at nanoscale
The overall objective of PC-II is to combine surface architectures of photoactivatable and membrane bilayer-destabilizing supramolecular materials with live cell imaging technologies to interrogate cargo transport and lipid dynamics at sites of plasma membrane (PM) permeabilization with nanoscale precision. Toward this end, supramolecular materials will be attached to planar or spherical surfaces via photocleavable linkers (Project 1). For optically controlled release of active compounds with sub-diffraction resolution, novel upconversion nano-triggers based on sensitized triplet-triplet annihilation will be developed (Project 4) that yield high-efficiency energy conversion under ambient conditions. These will be integrated into surface architectures to afford acute, light-induced and spatially confined PM permeabilization based on pulsed laser excitation. Gradient attractive optical forces will be used to support cargo transport (Project 2), while lipid scrambling and mobilization of membrane repair machinery to discrete PM areas with impaired barrier function will be monitored in real time using confocal spinning disc and TIRF microscopy (Project 3).
Project Cluster III
Tailored ultrathin macroporous substrates for correlative structural-functional analyses of protein complexes in membranes
In PC-III, we aim to establish correlative structural and functional analyses of insulated membrane protein complexes directly in native or biomimetic lipid bilayers by high-resolution microscopy techniques. For this purpose, we will develop membrane-macroporous substrate composites (MMSCs) consisting of a planar membrane deposited on a tailormade ultrathin substrate containing arrays of macropores with diameters from ~50 nm to a few 100 nm and thin enough to be compatible with cryoEM or near-field optical microscopy. The MMSCs will contain large numbers of unsupported membrane segments with minimized curvature, within which individual membrane-embedded protein complexes will be located. MMSCs will enable correlative structural-functional analyses of protein complexes by fluorescence microscopy and cryoEM, allowing monitoring protein conformational changes, protein interactions and complex assembly as well as protein-mediated membrane permeation by using the macropores as containers. Ultimately, we aim to implement MMSCs for correlative microscopy. To this end, Project 1 will devise a portfolio of MMSCs as a platform for combined structural and functional analysis of protein complexes in native and biomimetic membrane environments. Project 2 will interface appositely designed MMSCs with advanced fluorescence microscopy for the correlative structural and functional analysis of membrane pore formation by Gasdermin (GSDM) proteins in the plasma membranes of living cells. Project 3 will exploit MMSCs to produce free-standing lipid bilayers, in which the conformations and dynamics of ABC transporters will be analysed by cryoEM and, perspectively, correlative single-molecule FRET microscopy (smFRET).
Project Cluster IV
Carbon nanomaterial-supported membranes for optical and electrical interrogation of membrane fusion machineries
In PC-IV, we will exploit the unique electrical and optical properties of CNMs to explore the structural dynamics of the entire mem-brane fusion machinery on freely diffusing membranes at different stages of their action. We will develop membrane model systems on CNMs for functional reconstitution of the membrane-bound Ypt7 and HOPS for multicolour GIET measurements (Project 1). We will engineer a graphene-based FET (GFET) device for label-free detecting the structural dynamics of individual HOPS complexes upon vesicle tethering and fusion (Project 2). Expression, reconstitution and functional assays of the entire membrane fusion machinery with the membrane environments will be achieved for interfacing with the CNMs to resolve how the fusion machinery is organized and cooperates during fusion (Project 3), which can eventually be analysed by cryoEM.
