Journal
ADVANCED BIOLOGY
Volume -, Issue -, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/adbi.202300428
Keywords
channelrhodopsin-2; optogenetics; subcellular targeting; tandem repeats
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This study constructs three subcellularly-targeted optogenetic actuators based on mucin tandem repeats, and evaluates their expression and transfection efficiencies in different cell types. The results show that these actuators can be localized in both epithelial cells and neurons, opening up new possibilities for optogenetic manipulation in epithelial processes.
Optogenetics is a powerful approach in neuroscience research. However, other tissues of the body may benefit from controlled ion currents and neuroscience may benefit from more precise optogenetic expression. The present work constructs three subcellularly-targeted optogenetic actuators based on the channelrhodopsin ChR2-XXL, utilizing 5, 10, or 15 tandem repeats (TR) from mucin as N-terminal targeting motifs and evaluates expression in several polarized and non-polarized cell types. The modified channelrhodopsin maintains its electrophysiological properties, which can be used to produce continuous membrane depolarization, despite the expected size of the repeats. This work then shows that these actuators are subcellularly localized in polarized cells. In polarized epithelial cells, all three actuators localize to just the lateral membrane. The TR-tagged constructs also express subcellularly in cortical neurons, where TR5-ChR2XXL and TR10-ChR2XXL mainly target the somatodendrites. Moreover, the transfection efficiencies are shown to be dependent on cell type and tandem repeat length. Overall, this work verifies that the targeting motifs from epithelial cells can be used to localize optogenetic actuators in both epithelia and neurons, opening epithelia processes to optogenetic manipulation and providing new possibilities to target optogenetic tools. This work shows that tandem repeats from mucin can target the expression of optogenetic channels within the plasma membrane of multiple cell types. The constructs show the suitability of optogenetics in polarized epithelia. Tandem repeat targeting does not follow the typical apical-basal versus axonal-somatodendritic relationship for targeting constructs.image
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