high-throughput-imaging-analysis-workflow-for-evaluating-skin-cell

High-throughput Imaging and Analysis Workflow for Evaluating Skin Cell Phenotypes and Proliferation States in Tissue Samples

Skin injuries initiate a complex regenerative cascade that engages various cell types. Amongst those, peripheral glial cells play a critical role in ...

University of Zurich

The association of biomolecules is the elementary event of communication in biology. Most mechanistic information of how the interactions between binding partners form or break is, however, hidden in the transition paths, the very short parts of the molecular trajectories from the encounter of the two molecules to the formation of a stable complex. Here we use single-molecule spectroscopy to measure the transition path times for the association of two intrinsically disordered proteins that form a folded dimer upon binding. The results reveal the formation of a metastable encounter complex that is electrostatically favored and transits to the final bound state within tens of microseconds. Such measurements thus open a new window into the microscopic events governing biomolecular interactions.

Transition path times of coupled folding and binding reveal the formation of an encounter complex.

A bottleneck in many studies utilizing single-molecule Förster resonance energy transfer is the attainable photon count rate, as it determines the temporal resolution of the experiment. As many biologically relevant processes occur on time scales that are hardly accessible with currently achievable photon count rates, there has been considerable effort to find strategies to increase the stability and brightness of fluorescent dyes. Here, we use DNA nanoantennas to drastically increase the achievable photon count rates and observe fast biomolecular dynamics in the small volume between two plasmonic nanoparticles. As a proof of concept, we observe the coupled folding and binding of two intrinsically disordered proteins, which form transient encounter complexes with lifetimes on the order of 100 μs. To test the limits of our approach, we also investigated the hybridization of a short single-stranded DNA to its complementary counterpart, revealing a transition path time of 17 μs at photon count rates of around 10 MHz, which is an order-of-magnitude improvement compared to the state of the art. Concomitantly, the photostability was increased, enabling many seconds long megahertz fluorescence time traces. Due to the modular nature of the DNA origami method, this platform can be adapted to a broad range of biomolecules, providing a promising approach to study previously unobservable ultrafast biophysical processes.

Single-Molecule FRET at 10 MHz Count Rates.

Pulmonary conventional dendritic cells (cDCs) are functionally and phenotypically heterogeneous antigen-presenting cells essential for orchestrating adaptive immune responses in the lung. Here, we define a cell-intrinsic role for granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling in the development of a CD301b subset of terminally differentiated cDC2s, in addition to CD103XCR1 cDC1s. Unbiased single-cell transcriptomic profiling of CD11c cells identified both immature and differentiated lung cDC populations. GM-CSF deficiency disrupted antiapoptotic Bcl2a1 up-regulation and impaired progression to the CD301b transcriptional state. Despite the positioning of CD301b cDC2s in lymphoid cell-rich adventitial cuff areas, hematopoietic GM-CSF was dispensable for their development. Instead, alveolar epithelial type 2 cell-derived GM-CSF was required for CD301b cDC2 formation and pulmonary type 2 immune responses, highlighting the central role of GM-CSF signaling in shaping the pulmonary myeloid landscape.

GM-CSF derived from alveolar type 2 cells promotes CD301b cDC2 generation and allergic airway inflammation.

Signal transducer and activator of transcription (STAT) is a family of key transcriptional regulators in immune, epithelial and mesenchymal cells. Aberrant STAT activity is associated with malignancy, autoimmunity and immunodeficiency. The STAT signaling pathways are very attractive drug targets; however, validated tools to monitor real-time activation of STATs are lacking. Here, we developed a class of highly sensitive genetically encoded STAT biosensors, termed STATeLights, which allowed direct and continuous detection of STAT activity in live cells with high spatiotemporal resolution. Using human STAT5A, we demonstrate the versatility of STATeLight5A to quantify the activation of wild-type STAT5 versus disease-associated STAT5 mutants and to precisely select compounds targeting the STAT5 signaling pathway. Moreover, STATeLight5A also facilitated real-time tracking of STAT5 activation in human primary CD4 T cells. Collectively, our biosensors open up unprecedented possibilities of studying STAT biology and druggability in various cellular contexts.

Article	Year
Transition path times of coupled folding and binding reveal the formation of an encounter complex. Nature communications\| PubMed ID: 30413694	2018
Single-Molecule FRET at 10 MHz Count Rates. Journal of the American Chemical Society\| PubMed ID: 38266173	2024
GM-CSF derived from alveolar type 2 cells promotes CD301b cDC2 generation and allergic airway inflammation. Science immunology\| PubMed ID: 40779647	2025
Real-time visualization of STAT activation in live cells using genetically encoded biosensors. Nature chemical biology\| PubMed ID: 40890507	2025

Flurin Sturzenegger

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