International Journal of Aquaculture, 2024, Vol.14, No.1, 37-39 http://www.aquapublisher.com/index.php/ija 38 Figure 1 shows the process of Wnt5b-Ror2 complex transport from producer cells to recipient cells through cytonemes, and analyzes this transport mechanism in detail. Figures 1a and 1b show that Wnt5a/b is mainly localized on the cell processes of zebrafish fibroblasts (PAC2 cells), and the cell processes with Wnt5a/b are significantly longer. This result shows that the presence of Wnt5a/b in cell protrusions is directly related to the length of these protrusions, suggesting its important role in the signal transmission process. Figures 1c to 1e further demonstrate the colocalization of Ror2 and Wnt5b on cell protrusions, and staining with anti-Ror2 and anti-Wnt5a/b antibodies confirmed the co-localization of Ror2 and Wnt5a/b on these protrusions. The experiments in Figures 1h to 1k demonstrate the colocalization of Wnt5b-GFP and Ror2-GFP on cell protrusions and their endocytosis in the receiving cell by fluorescent labeling and treatment with a dynasore (Dyn) inhibitor. These results illustrate that the Wnt5b-Ror2 complex remains intact during transport through cell protrusions and that endocytosis is dependent on dynein. This illustration provides an intuitive model of how the Wnt5b and Ror2 complexes carry out cell-to-cell transmission and signaling through cell protrusions. Figure 1 Wnt5b-Ror2 complexes are transported from the producing cells to the receiving cells via cytonemes Figure 4 demonstrates the paracrine signaling mechanism mediated by the Wnt5b-Ror2 complex through cell processes through experimental data, revealing the key role of this process in JNK signal activation. Figure 4a shows that in wild-type zebrafish embryos, clones expressing Wnt5b-Ror2 were able to significantly activate JNK signaling, demonstrating the effectiveness of the Wnt5b-Ror2 complex. Figure 4b quantifies the relative intensity of JNK signaling activated by different doses of Wnt5b-Ror2 clones within five cell rows, and the results show that the activation of JNK signaling is significantly enhanced as the Wnt5b-Ror2 dose increases. Figures 4c and 4d demonstrate the effect of IRSp534K mutant on JNK signaling activation. IRSp534K is a mutant that inhibits cell protrusion formation. These experimental results indicate that cell protrusions are not only a key channel for Wnt5b-Ror2 complex transmission, but also play an important role in the regulation of signal intensity. 3 Breakthrough in Intercellular Signaling: Cell Protrusions Drive Wnt5b-Ror2 Transport in Zebrafish Embryos By revealing the cell protrusion-mediated transport mechanism of the Wnt5b-Ror2 complex in zebrafish embryos, this study makes a significant contribution to the field of intercellular signaling and challenges the traditional diffusion model of signaling molecules. This study demonstrates the critical role of cell protrusions in the precise delivery of signaling molecules, highlighting the mechanism by which producer cells deliver active ligand-receptor complexes directly to receiving cells via cell protrusions.
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