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Monitoring the Response of Multiple Signal Network Components to Acute Chemo-Optogenetic Perturbations in Living Cells |
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| Authors: | Dr Manuela Kowalczyk Dominic Kamps Prof Yaowen Wu Leif Dehmelt Prof Perihan Nalbant |
| Affiliation: | 1. Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany
These authors contributed equally to this work.;2. Department of Chemistry and Chemical Biology and Department of Systemic Cell Biology, TU Dortmund University and Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany These authors contributed equally to this work.;3. Department of Chemistry, Umeå Centre for Microbial Research, Umeå University, 901 87 Umeå, Sweden;4. Department of Chemistry and Chemical Biology and Department of Systemic Cell Biology, TU Dortmund University and Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany;5. Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany |
| Abstract: | Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy-based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo-optogenetic approach that is based on switch-like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback-controlled regulation in the cell contraction signal network that includes GEF-H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants. |
| Keywords: | optogenetics photocaging RhoA sensors signal networks |