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. 2023 Jul 25;80(8):228. doi: 10.1007/s00018-023-04876-8

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Field stimulation results in subnuclear redistribution of Jacob that correlates with enhanced [Ca²⁺]_n. a Schematic representation of pS180-Jacob-CREB PLA assay. b–c Five-minute treatment of hippocampal neurons with 4AP/bicuculline (2,5 mM/ 50 μM) results in dramatic association of pS180-Jacob with CREB, as revealed by significant increase in PLA intensity within the nucleus. Panel b depicts maximum projection confocal image of cultured hippocampal neurons (DIV16) incubated with anti-pJacob (rb) and anti-CREB (ms) antibodies and proceeded for proximity ligation followed by labeling with anti-MAP2 antibodies and DAPI; Scale bar = 10 μm, ***p < 0.001 by t test. d Timeline of the experiment. e Confocal time-lapse images of hippocampal neuron overexpressing Jacob-GFP before and after field stimulation (18’’@50 Hz) showed an increase in fluorescent intensity at the nuclear matrix and a decrease in intensity at the nuclear envelope upon stimulation. f Graph represents averaged intensities of Jacob-GFP (n = 6). The blue line indicates the trace of fluorescent intensity changes within the nuclear matrix upon field stimulation; the orange line indicates the trace of fluorescent intensity changes at the nuclear lamina. g–i Field stimulation (18 s@50 Hz) resulted in a simultaneous increase in nuclear calcium, reflecting the pattern of neuronal activity, and a rise in Jacob-CyRFP2 fluorescent intensity. g Confocal time-lapse images of hippocampal neuron overexpressing Jacob-CyRFP2 and loaded with Fluo-4 (AM). f Averaged normalized intensities of Jacob and Fluo-4 changes upon stimulation. i The plot indicates a high correlation between Jacob-CyRFP2 and Fluo-4 (AM) intensities changes upon LTP-like stimuli. Pearson r = 0.7997, p < 0.0001. j Graph represents induction of LTP with TBS in CA1 area of the hippocampus. k–l TB stimulation induces significant association of pS180-Jacob with CREB as revealed by pJacob-CREB PLA. l ^##p < 0.01, Nested t test m ^##p < 0.01, Nested t test, n–p TB stimulation results in dissociation of Jacob from LaminB1. o ^#p < 0.05, Nested t test. p ^#p < 0.05, Nested t test. q–r Neurons expressing Jacob at the nuclear lamina and neuronal nuclei characterized by enhanced expression of luciferase as compared to neurons expressing Jacob extranuclear. Schematic representation of the Activity-Responsive Element luciferase reporter (SARE-ArcMin-Luc) where SARE is fused upstream of TATA-containing sequence of the Arc. Panel q depicts maximum projection confocal images of cultured hippocampal neurons (DIV16) co-transfected with Jacob and SARE-ArcMin-Luc overnight followed by labeling with anti-Luciferase, anti-MAP2 antibodies and DAPI; scale bar = 10 μm, ***p < 0.001 by t test