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. 2018 Feb 13;8:2903. doi: 10.1038/s41598-018-20893-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Molecular determinants of GCAP1 distribution to rod outer segments in vivo. Wildtype and different mutant forms of GCAP1 were expressed as transient transgenes in the rods of GCAP1/GCAP2 knockout mice. Mosaicism results from the in vivo DNA electroporation method of transfection. GCAP1 WT (green signal in B,C) distributed 50:50% between the inner and outer segment compartments. K23D/GCAP1 (green signal, E,F) was retained at the inner segment, as if its distribution to rod outer segments was precluded. W94A/GCAP1 (green signal, H,I) reproduced the wildtype localization. G2A/GCAP1 (green signal, K,L) was retained at the inner segment. In red, rhodopsin mAb1D4 labels the rod outer segment layer (A,D,G,J and merged images). M. Percentage of GCAP1 signal at the outer segment compartment (from the combined signal at outer and inner segments). Mean values are indicated, with bars representing the standard error. Mean ± SEM were: WT (●) 51.30 ± 4.38, n = 18; K23D (▲) 9.48 ± 2.92, n = 15; W94A (▼) 45.58 ± 4.55, n = 11; G2A (■) 6.49 ± 3.73, n = 13. T-test was used to determine statistical significance versus WT. For G2A and K23D mutants, p-value < 0.0001. At least three injected animals per construct were analyzed, showing consistent results. The outer segment length may vary depending on the position of the acquired field-image on the retina section.