TY - JOUR
T1 - Optogenetic regulation of endogenous proteins
AU - Redchuk, Taras A.
AU - Karasev, Maksim M.
AU - Verkhusha, Polina V.
AU - Donnelly, Sara K.
AU - Hülsemann, Maren
AU - Virtanen, Jori
AU - Moore, Henna M.
AU - Vartiainen, Maria K.
AU - Hodgson, Louis
AU - Verkhusha, Vladislav V.
N1 - Funding Information:
We thank E. Omelina for the help with cloning, M. Affolter for the pcDNA3_NSlmb-vhhGFP4 plasmid (Addgene #35579), O. Pertz for the pLentiEKAR2G2 plasmid (Addgene #40178), J. Zhang for the pcDNA3-AktAR2 plasmid (Addgene #64932), R. Seger for the pGFP-ERK2 plasmid (Addgene #37145), and K. Svoboda for the mEGFP-HRAS plasmid (Addgene #18662). We also thank the Biomedicum Flow Cytometry Unit, Biomedicum Imaging Unit, and Light Microscopy Unit (all University of Helsinki) for the technical assistance. This work was supported by the grants GM122567, NS103573 (both to V.V.V.) and GM129098, GM132098 (both to L.H.) from the US National Institutes of Health, 322226 from the Academy of Finland (to V.V.V.), American Cancer Society Lee National Denim Day Postdoctoral Fellowship PF-15-135-01-CSM (to S.D.), Irma T. Hirschl Career Scientist Award (to L.H.), and by Jane and Aatos Erkko foundation (to M.K.V.).
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.
AB - Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.
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U2 - 10.1038/s41467-020-14460-4
DO - 10.1038/s41467-020-14460-4
M3 - Article
C2 - 32001718
AN - SCOPUS:85078710529
VL - 11
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 605
ER -