Application of modified fluorophore-assisted light inactivation technique in nervous system cell and explant cultures

Main Article Content

Kohtaro Takei

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.

Keywords: Light-mediated protein disruption, FALI, SELT-FALI, Organotypic culture

Article Details

How to Cite
TAKEI, Kohtaro. Application of modified fluorophore-assisted light inactivation technique in nervous system cell and explant cultures. Medical Research Archives, [S.l.], v. 6, n. 5, may 2018. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/1800>. Date accessed: 05 oct. 2024. doi: https://doi.org/10.18103/mra.v6i5.1800.
Section
Articles

References

1) Jay DG. Selective destrcution of protein function by choromophore-sdisted laser inactivation. Proc Natl Acad Sci USA. 1988;85:5454-5458.

2) Beck S, Sakurai T, Eustace BK, et al. Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins. Proteomics. 2002;2:247-255.

3) Eustace BK, Sakurai T, Stewart JK, et al. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nature Cell Biology. 2004;6:507-514.

4)Jay DG, Keshishian H. Laser inactivation of fasciculin I disrupts axon adhesion of grasshopper pioneer neurons. Nature. 1990;348:548-550.

5) Surrey T, Elowitz MB, Wolf PE, et al. Chromophore-assisted light inactivation and self-organization of microtubules and motors. Proc Natl Acad Sci USA. 1998;95:4293-4298.

6) Marek KW, Davis GW. Transgenically encoded protein photoinactivation (FlAsH-FALI) acute inactivation of synaptotagmin I. Neuron. 2002;36:805-813.

7) Tour O, Meijer RM, Zacharias DA, et al. Genetically targeted chromophore-assated light inactivqation. Nature Biotech. 2003;21: 505-1508.

8) Takemoto K, Matsuda T, McDougall M, et al. Chromophore-asssted light inactivation of Halo Tag fusion proteins labeled with eosin in living cells. ACS Chem Biol. 2011;6:401-406.

9) Sano Y, Watanabe W, Matsunaga S. Choromophore-assisted laser inactivation-towards a spatiotemporal-functional analysis of proteins, and the ablation of chromatin, organelle and cell function. J. Cell Sci. 2014;127:1621-1629.

10) Liao JC, Roider J, Jay DG. Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals. Proc Natl Acad Sci USA. 1994;91:2659-2663.

11) Chen H, Chedotal A, He ZG, et al. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron. 1997;19:547-559.

12) Takahashi T, Nakamura F, Jin Z, et al. Semaphorins A and E act as antagonists of neuropilin-1 and agonists of neuropilin-2 receptors. Nature Neurosci. 1998;1:487-493.

13) Messersmith EK, Leonardo ED, Shatz CJ, et al. Semaphorin-III Ccan function as a selective chemorepellent to pattern sensory projections in the spinal cord. Neuron. 1995;14:949-959.

14) Sato Y, Hirata T, Ogawa M, et al. Requirement for early-generated neurons recognized by monoclonal antibody Lot1 in the formation of lateral olfactory tract. J Neurosci. 1998;18:7800-7810.

15) Olsen TW, Sternberg P, Reed RL, et al. A model for light toxicity of cultured human retinal pigment epithelium. Graefes Arch Clini Exp Ophthal. 1997;235: 11-117.

16) Kappus H. Oxidative stress in chemical toxixity. Arch Toxicol, 1987;60:144-149.