B4.1: Small lock-and-key elements to track and manipulate receptors in time and space

The PhD student will explore wavelength-addressable optochemical tools for the spatiotemporal labeling and assembly of proteins. Novel small lock-and-key elements will be developed in order to site-specifically label, track and manipulate receptor proteins in time and space. The photophysical properties (e.g. uncaging quantum yield, two-photon cross-section) of the light-sensitive moieties will be analyzed and optimized in close collaboration with A2.1. Further performance tests of the newly developed PA-trisNTAs will be undertaken at functionalized interfaces (surface plasmon resonance, TIRFM, etc.). By strategic positioning of black-hole quenchers, photo-cleavage will cause fluorescence dequenching and allow high affinity labeling upon illumination for super-resolution microscopy (in collaboration with B5.1).

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B4.2: In-situ assembly of receptors triggered by light

This project aims at introducing the newly designed PA-trisNTAs into cell surface receptors by bioorthogonal labeling. Based on the self-inactivation, the PA-trisNTA and His-tagged receptors will interact only upon light activation, which can be performed in dual or triple color mode by LSM or TIRF microscopy. By the generation of protein binding regions upon illumination, the process of iterative protein binding and sequential clustering of membrane receptors will be examined. The project will take advantage of a range of receptors fused to a fluorescent reporter protein and advanced microscopy in collaboration with B5.1.

The PhD student will be able to control receptor clustering in vitro and later in vivo. The lateral organization of membrane receptors will be realized in giant unilamellar vesicles (GUV), solid supported membranes, as well as the plasma membrane of living cells. The receptor mobility before and after photo-activation will be analyzed with fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single-particle tracking in combination with photo-switchable fluorophores (in collaboration with B5.1).

Light induced receptor clustering in time and space. A) Chemical structure of PA-trisNTA and examples of wavelength selective caging groups. B) PA-trisNTA is covalently attached to receptors on living cells. This provides interaction of orthogonal receptor pairs triggered by light.

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B4.3: 3D organization and manipulation of proteins by light

This project will focus on the in-situ assembly of biomolecules in 3D scaffolds. Laser scanning lithography combined with light-triggered protein assembly offers a powerful tool to organize protein gradients and clustering for a broad range of applications. The PhD student will employ the site-specific and spatiotemporal organization of proteins as well as the sequential protein clustering in 3D scaffolds. For elaborated applications, the photophysical properties, like wavelength selective-activation and two-photon accessibility will be systematically optimized (with A2.2 and A3.2). The process of iterative binding and sequential protein clustering of proteins, including the fabrication of lateral protein gradients will then be examined. Mask-patterned illumination and laser lithography will be employed to write in-situ regions and protein gradients.

Figure: In-situ protein assembly by optochemical tools. A) Comparison of one and two photon activation in hydrogels by light (a) and three dimensional protein assembly by two photon laser-scanning microscopy (b).

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