Primed Conversion

The PANTAZIS LAB captures the chemical and mechanical dynamics 

of development and disease by using cutting-edge approaches to live imaging.

The fields of systems biology and recently precision medicine were made possible by genomics as well as complementary high-throughput approaches such as microarrays and proteomics. Using genome-wide, high-throughput -omics analyses, structures of biological circuits are increasingly being uncovered. These approaches give significant insight into the components and interactions that comprise biological networks on an unprecedented scale in several organisms. However, rough static models are relatively coarse, error prone, and provide limited information of biological dynamics during development and disease.

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Live imaging offers the unique advantage of observing biological processes with high spatiotemporal resolution in whole organisms, offering a path to more refined, quantitative dynamic models. The introduction of advanced imaging tools and automated instrumentation is the main focus of my laboratory, which will enable us to apply imaging for both hypothesis-driven research and high-throughput analysis.

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Recent major research contributions 

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Since establishing the lab, the aim of Dr Pantazis' research activity was to develop advanced imaging technologies (probes, imaging modality, and quantitative analysis; see Fig. 1) to establish an effective acquisition and interpretation workflow i) for the mechanistic analysis of biological systems in animal models such as mouse and zebrafish and ii) for the use in novel diagnostic and therapeutic strategies.

 

 
To get more insight into the elaborate cell and protein dynamics that underlie development and disease(1), the lab introduced a unique optical mechanism, primed conversion, where dual-wavelength illumination results in pronounced photoconversion of photoconvertible fluorescent proteins (pcFPs)(2-4). As two-photon-based photoconversion is extremely inefficient, primed conversion is the only way to precisely photoconvert in 3D pcFPs for real-time in vivo studies aiming to unravel complex structural and dynamic information. Using confined primed conversion, we revealed the complex anatomy of individual neurons packed between neighboring cells in zebrafish(2,5). The combination of primed conversion and a spatial drift correction algorithm, primed Track, allowed us to accomplish high-fidelity volumetric lineage tracing in mouse pre-implantation embryos(6,7). Primed conversion has also been successfully extended to manipulate the pcFP-based optogenetic effector, photocleavable protein (PhoCl). PhoCl spontaneously dissociates into two fragments after light-induced cleavage of a specific bond in the protein backbone, opening the path to transcriptional manipulation of cells in vivo at single cells resolution(8). Using engineered optimized primed convertible FPs (pr-FPs), we (and others) have applied primed conversion also in non-toxic single molecule dynamic analysis using super-resolution imaging(9).

 
Throughout an organism’s lifetime, cell mechanosensation (i.e., the ability to perceive and respond to mechanical stimuli in the form of shear stress, tension, or compression) is essential in a myriad of developmental, physiological, and pathophysiological processes including embryogenesis, homeostasis, metastasis, and wound healing. To investigate how physical forces and changes in mechanical properties of cells contribute to development and disease, we designed the fluorescent reporter GenEPi for visualizing dynamics and mechanical stimuli of Piezo1, an essential mechanosensitive ion channel found in plants and animals(10). We show that the intensiometric, genetically-encoded reporter GenEPi has high specificity and spatiotemporal resolution for Piezo1-dependent mechanical stimuli, exemplified by resolving repetitive mechanical stimuli of spontaneously contracting cardiomyocytes within microtissues and revealing mobile and functionally dynamic Piezo1 clusters in the plasma membrane using time-lapse TIRF imaging. GenEPi is an ideal tool to elucidate the full extent to which mechanical signals, and more specifically Piezo1 channels, regulate development, physiology, and disease(11).

 





Previously, we introduced inorganic second harmonic generating (SHG) nanocrystals, SHG nanoprobes, as a class of imaging probes that can be used for in vivo imaging(12-15). Given that SHG imaging employs near-infrared (NIR) incident light for contrast generation, SHG nanoprobes can be utilized for deep tissue imaging. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and their signal does not saturate with increasing illumination intensity, ensuring high probe sensitivity(16-18). To create a foundation for safe SHG nanoprobe-based clinical imaging, we generated bioharmonophores as a novel class of imaging probes that retain all the photophysical advantages of previously introduced inorganic SHG nanoprobes. Because bioharmonophores consist of a biodegradable peptide core and a polymer shell, they can be metabolized within cells, which render them ideal contrast agents for clinical imaging applications. The straightforward implementation of robust functionalization strategies and a sufficiently high metabolic stability in vivo allowed us to target bioharmonophores with high detection sensitivity to individual tumor cells in vivo(19,20).

GenEPi - Force Sensor

Bioharmonophores

 
 
Screenshot 2021-12-12 at 3.21.46 pm.png

REFERENCES​

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  1. Plachta, N., Bollenbach, T., Pease, S., Fraser, S. E. & Pantazis, P. Oct4 kinetics predict cell lineage patterning in the early mammalian embryo. Nat Cell Biol 13, 117-123, doi:10.1038/ncb2154 (2011).

  2. Dempsey, W. P. et al. In vivo single-cell labeling by confined primed conversion. Nat Methods 12, 645-648, doi:10.1038/nmeth.3405 (2015).

  3. Mohr, M. A., Argast, P. & Pantazis, P. Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins. Nat Protoc 11, 2419-2431, doi:10.1038/nprot.2016.134 (2016).

  4. Mohr, M. A. & Pantazis, P. Primed Conversion: The New Kid on the Block for Photoconversion. Chemistry 24, 8268-8274, doi:10.1002/chem.201705651 (2018).

  5. Mohr, M. A. & Pantazis, P. Single neuron morphology in vivo with confined primed conversion. Methods in cell biology 133, 125-138, doi:10.1016/bs.mcb.2015.12.005 (2016).

  6. Welling, M. et al. Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins. eLife 8, doi:10.7554/eLife.44491 (2019).

  7. Welling, M., Kalyviotis, K. & Pantazis, P. P. Primed Track: Reliable Volumetric Single-cell Tracking and Lineage Tracing of Living Specimen with Dual-labeling Approaches. Bio-Protocol 10, doi:10.21769/BioProtoc.3645 (2020).

  8. Zhang, W. et al. Optogenetic control with a photocleavable protein, PhoCl. Nat Methods 14, 391-394, doi:10.1038/nmeth.4222 (2017).

  9. Mohr, M. A. et al. Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion. Angewandte Chemie (International ed. in English) 56, 11628-11633, doi:10.1002/anie.201706121 (2017).

  10. Yaganoglu, S. et al. GenEPi: Piezo1-based fluorescent reporter for visualizing mechanical stimuli with high spatiotemporal resolution. bioRxiv, 702423, doi:10.1101/702423 (2019).

  11. Landhuis, E. Seven technologies to watch in 2021. Nature 589, 630-632, doi:10.1038/d41586-021-00191-z (2021).

  12. Pantazis, P., Pu, Y., Psaltis, D. & Fraser, S. Second Harmonic Generating (SHG) Nanoprobes: a New Tool for Biomedical Imaging. Proc Spie 7183, doi:Unsp 71831p10.1117/12.808434 (2009).

  13. Pantazis, P., Maloney, J., Wu, D. & Fraser, S. E. Second harmonic generating (SHG) nanoprobes for in vivo imaging. Proc Natl Acad Sci U S A 107, 14535-14540, doi:10.1073/pnas.1004748107 (2010).

  14. Cohen, B. E. Beyond fluorescence. Nature 467, 407-408, doi:10.1038/467407a (2010).

  15. Culic-Viskota, J., Dempsey, W. P., Fraser, S. E. & Pantazis, P. Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish. Nat Protoc 7, 1618-1633, doi:10.1038/nprot.2012.087 (2012).

  16. Dempsey, W. P., Fraser, S. E. & Pantazis, P. SHG nanoprobes: advancing harmonic imaging in biology. BioEssays : news and reviews in molecular, cellular and developmental biology 34, 351-360, doi:10.1002/bies.201100106 (2012).

  17. Pantazis, P. & Supatto, W. Advances in whole-embryo imaging: a quantitative transition is underway. Nat Rev Mol Cell Biol 15, 327-339, doi:10.1038/nrm3786 (2014).

  18. Sugiyama, N., Sonay, A. Y., Tussiwand, R., Cohen, B. E. & Pantazis, P. Effective Labeling of Primary Somatic Stem Cells with BaTiO3 Nanocrystals for Second Harmonic Generation Imaging. Small 14, doi:10.1002/smll.201703386 (2018).

  19. Sonay, A. Y. & Pantazis, P. in Clinical and Preclinical Optical Diagnostics Vol. 10411   (2017).

  20. Sonay, A. Y. et al. Biodegradable Harmonophores for Targeted High-Resolution In Vivo Tumor Imaging. ACS Nano, doi:10.1021/acsnano.0c10634 (2021).

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P A N T A Z I S  L A B.