Multifunctional molecular probes for fluorescence imaging of cell membranes – Cellmemprobes

The project is composed of five tasks. Task 1 is project coordination. In Task 2 we will synthesize new fluorescent membrane probes using methods of organic synthesis and bioconjugation. The typical fluorescent membrane probe will be composed of a fluorophore and an amphiphilic anchor group. The receptor-specific membrane probe will bear a specific ligand through an appropriate spacer. In Task 3, fluorescence response of the probes to lipid order will be evaluated on large unilamellar vesicles and giant vesicles using fluorescence spectroscopy and microscopy. In Task 4, the probes selected in Task 3 will be further evaluated in living cells in terms of binding to plasma membrane, internalization and cytotoxicity. In Task 5 we will develop applications of the probes for (1) imaging lipid order and microdomains and for (2) receptor recognition and probing its lipid surrounding. The probes will be tested in cell lines expressing corresponding receptors (integrins, oxytocin receptor, etc) in order to evaluate their turn-on response to the receptors and to detect lipid order around these receptors.

(1) A variety of new fluorescent membrane probes were developed: (a) two-color probes for lipid order and apoptosis ; (b) bright and photo-stable blue probes for multi-color imaging ; (c) far-red probes based on squaraine with exceptional brightness for in vitro and in vivo imaging. (2) We obtained a tool-kit of Nile Red-based probes with FRET quencher that can specifically target and identify separate phases in cell membranes, providing new evidences of lipid rafts. (3) Innovative fluorogenic probes for membrane receptors were obtained based on Nile Red, squaraine monomer and dimers.

(1) We expect that the new probes will be useful for a broad community of scientists working on membrane biology, in particular to study membrane receptors and membrane lipid organization. Moreover, the probes for receptor detection may find applications in drug screening and clinical diagnostics for imaging of the target cells. (2) We expect to obtain important biological insights on the structure, dynamics and functions of membrane domains and provide new evidences for their interaction with membrane proteins.

New membrane probes for lipid domains and multicolor imaging:
(1) Kreder et al, RSC Adv., DOI: 10.1039/C4RA16225K. (2) Kreder et al, ACS Chem. Biol. (accepted). (3) Niko et al, Chem. Eur. J. 2014, 20, 16473. (4) Darwich et al, RSC Adv., 2014, 4, 2014, 8481. (5) Klymchenko & Kreder Chem. Biol., 2014, 21, 97. (6) Darwich et al, MAF J. 2013, 1, 025002. (7) Darwich et al, Biochm. Biphys. Acta 2012, 1818, 3048. (8) Kucherak et al, Phys. Chem. Chem. Phys. 2012, 14, 2292.

Receptor-specific turn-on probes:
(1) Karpenko et al, Chem. Commun., 2015, 51, 2960. (2) Karpenko et al, J. Am. Chem. Soc. 2015, 137, 405. (3) Karpenko, Kreder, et al, ChemBioChem, 2014, 15, 359.

Applications of membrane probes in collaboration: 8 articles.

Fluorescence bioimaging is a rapidly expanding research area because it opens possibilities for non-invasive detection techniques, which are of high interest both in research and clinical diagnostics. Fluorescence imaging requires fluorescent tools, so-called fluorescent probes. One of the most attractive targets in cellular imaging is plasma membranes. The design of new membrane probes for broad applications in biology requires their multifunctionality, which differentiates them from classical markers of lipid membranes. The present project is aimed to develop fluorescent membrane probes featuring the following functions. The primary one is sensitivity to membrane properties, particularly to lipid order. The latter is connected with the phenomenon of membrane domains (rafts), which are believed to play a key role in the activity of membrane proteins. This sensitivity can be achieved by the use of environment-sensitive dyes, which can monitor properties of their surrounding by change in their fluorescence intensity and color. The second desired function of membrane probes is specific targeting of cell plasma membranes and notably the outer membrane leaflet, accompanied by the possibility to “turn on” the probe fluorescence on membrane binding. The specific labeling of the membrane outer leaflet is important because it is enriched with lipids forming raft domains. Another important aspect is to design functionalized membrane probes able to target particular membrane receptors. These probes could allow both simple detection of the target cells and new applications, such as monitoring lipid order selectively in the receptor surrounding. Our group had been strongly involved in the field of membrane probe development (>25 articles and a licensed patent) and the obtained results provide a strong background for successful development of the desired multifunctional probes. The present project is composed of five Tasks. The first Task is project coordination. In Task 2 we will synthesize new membrane probes by coupling environment-sensitive fluorophores or a quencher with appropriate amphiphile groups. The obtained probes will feature improved fluorescence properties, selectivity to the outer leaflet with minimal flip-flop, efficient membrane binding and better solubility. To achieve probe specificity to a given membrane receptor, they will be further conjugated with a specific ligand. After detailed characterization of the obtained probes in solutions and model lipid vesicles (Task 3), they will be further characterized in cells (Task 4) in order to examine their binding to cell plasma membrane, flip-flop between the leaflets, cell internalization, and cytotoxicity. The final Task 5 deals with two biological applications of selected probes. First one is imaging of ordered lipid domains in cells using advanced fluorescence microscopy setups (single molecule and super-resolution microscopy, such as STED and PALM). The second application is receptor recognition by membrane probes and monitoring the lipid order in the receptor surrounding. The consortium is a highly interdisciplinary team composed of seven young researchers of complementary expertise. Notably three of them were awarded in 2010 for scientific excellence. The results of the project will be published in top-ranked journals, while the best membrane probes will be patented and commercialized. We expect that the new probes will find broad application in cellular research, particularly on membrane rafts and membrane proteins. Moreover, they may find applications in clinical diagnostics for imaging of the target cells.