Biochemie, Chemie und Pharmazie
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The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.
Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors
Maintenance of the bacterial homeostasis initially emanates from interactions between proteins and the bacterial nucleoid. Investigating their spatial correlation requires high spatial resolution, especially in tiny, highly confined and crowded bacterial cells. Here, we present super-resolution microscopy using a palette of fluorescent labels that bind transiently to either the membrane or the nucleoid of fixed E. coli cells. The presented labels are easily applicable, versatile and allow long-term single-molecule super-resolution imaging independent of photobleaching. The different spectral properties allow for multiplexed imaging in combination with other localisation-based super-resolution imaging techniques. As examples for applications, we demonstrate correlated super-resolution imaging of the bacterial nucleoid with the position of genetic loci, of nascent DNA in correlation to the entire nucleoid, and of the nucleoid of metabolically arrested cells. We furthermore show that DNA- and membrane-targeting labels can be combined with photoactivatable fluorescent proteins and visualise the nano-scale distribution of RNA polymerase relative to the nucleoid in drug-treated E. coli cells.
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.
Super-resolution fluorescence microscopy revolutionizes cell biology research and provides novel insights on how proteins are organized at the nanoscale and in the cellular context. In order to extract a maximum of information, specialized tools for image analysis are necessary. Here, we introduce the LocAlization Microscopy Analyzer (LAMA), a comprehensive software tool that extracts quantitative information from single-molecule super-resolution imaging data. LAMA allows characterizing cellular structures by their size, shape, intensity, distribution, as well as the degree of colocalization with other structures. LAMA is freely available, platform-independent and designed to provide direct access to individual analysis of super-resolution data.