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Institut
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and mesoscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a chelator nanotool. Due to the multivalent interaction, we observed a dynamic exchange in the microscale confined regions. Fast Y2R enrichment in clustered areas triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading, and migration. We revealed that the cell response to ligand-induced activation was amplified when cells were pre-clustered by the nanotool. Ligand-independent signaling by clustering differed from ligand-induced activation in the binding of arrestin-3 as downstream effector, which was recruited to the confined regions only in the presence of the ligand. This approach enables in situ clustering of membrane receptors and raises the possibility to explore different modalities of receptor activation.
Internalin B–mediated activation of the membrane-bound receptor tyrosine kinase MET is accompanied by a change in receptor mobility. Conversely, it should be possible to infer from receptor mobility whether a cell has been treated with internalin B. Here, we propose a method based on hidden Markov modeling and explainable artificial intelligence that machine-learns the key differences in MET mobility between internalin B–treated and –untreated cells from single-particle tracking data. Our method assigns receptor mobility to three diffusion modes (immobile, slow, and fast). It discriminates between internalin B–treated and –untreated cells with a balanced accuracy of >99% and identifies three parameters that are most affected by internalin B treatment: a decrease in the mobility of slow molecules (1) and a depopulation of the fast mode (2) caused by an increased transition of fast molecules to the slow mode (3). Our approach is based entirely on free software and is readily applicable to the analysis of other membrane receptors.
Membrane receptor clustering is fundamental to cell–cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we train the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-colour super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule imaging modality to enable fast single-molecule super-resolution microscopy.
HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.
Highlights
HER2 exhibits heterogeneous motion in the plasma membrane
The fraction of immobile HER2 correlates with phosphorylation levels
Diffusion properties serve as proxies for HER2 activation
HER2 exhibits ligand-specific activation strength and temporal profiles