Application of modified fluorophore-assisted light inactivation technique in nervous system cell and explant cultures
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Abstract
Methods of molecular targeting are powerful tools for functional genomics studies. To analyze the biological function of the molecule, acute and temporally restricted inactivation of the targeted molecule is critical. We developed a simple experimental method of protein disruption in living nervous systems. We employed a fluorophore-assisted light inactivation (FALI) technique that uses light irradiation to produce photogenerated singlet oxygen free radical damage that is directed at targeted proteins through chromophore-conjugated antibodies. In order to apply the FALI technique to culture systems over a long period of time, we established a simple and easy-to-use long-term FALI (SELT-FALI) by modifying the original FALI to utilize weak blue light. To address the efficacy of the SELT-FALI technique, we used Neuropilin-2 (Nrp2), a receptor for a repulsive axon guidance molecule of Semaphorin-3F (Sema3F) as a test target molecule. We examined the effect of SELT-FALI of Nrp2 by assessing specific Sema3F-Nrp2 binding in various culture systems. SELT-FALI of Nrp2 resulted in complete inhibition of Sema3F binding to Nrp2 expressed on COS7 cells. This FALI efficacy was dependent on irradiated light power. SELT-FALI of Nrp2 decreased Sema3F-induced growth cone collapse in mouse sympathetic ganglion culture, and also resulted in a significant loss of repulsive response toward Sema3F in a collagen gel 3D culture system. Furthermore, continuous FALI of Nrp2 for 24 hours in developing neural tissue resulted in a significant reduction of Sema3F binding to Nrp2 expressing lateral olfactory tract (LOT) in organotypic culture of mouse telencephalon without any detrimental effects on LOT development. Thus, the FALI technique can be used for protein disruption with temporal resolution. Finally, we applied the SELT-FALI technique for functional screening of key molecules in LOT development, which resulted in the discovery of a novel functional molecule that functions in LOT formation, and this molecule was termed the lateral olfactory tract usher substance (LOTUS). This methodology can be used for functional screening of key molecules in a variety of culture systems.
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