Journal: Nature Communications

Article Title: Mechanochemical mechanism underlying intercellular Ca 2+ wave propagation and its crucial role in apoptotic cell extrusion

doi: 10.1038/s41467-025-65474-9

Figure Lengend Snippet: a LUT color-coded images of GCaMP7 and Lifeact-GFP in embryos treated with DMSO (control), Gd 3+ , GsMTx4, NS8593, Yoda1, XestoC, 2-APB, BTP-2, αGA, CBX, JTE013, SKI-II, Apyrase, Suramin, and αGA + Apyrase; and WT ( trpc1 +/+ ), heterozygous ( trpc1 +/- ), and homozygous ( trpc1 - /- ) embryos injected with or without piezo 1-MO, and iptr 1 a + iptr 1 b at 0 s (left panels) or 30 s (right panels) after irradiation with a 150 nJ/pulse femtosecond laser through a 40×/0.8 NA objective lens. White dotted lines and red asterisks indicate the Ca 2+ wave propagation area and laser focal point, respectively. The effectiveness of apyrase or suramin treatment on Ca 2+ wave propagation in zebrafish embryonic epithelia was shown in Supplementary Fig. . Scale bar: 50 µm. b The propagation area of the Ca 2+ wave. n = 16, 9, 13, 8, 8, 11, 10, 10, 13, 12, 8, 11, 9, 11, and 8 embryos. c Effects of trpc1 knockout and/or piezo 1 knockdown on the expansion area of the Ca 2+ wave. n = 12, 19, 6, 9, 17, and 6 embryos. d Effects of itpr 1 a + itpr 1 b knockdown on the expansion area of the Ca 2+ wave. Control-MO: n = 5 and itpr 1 a -MO plus itpr 1 b -MO: n = 12 embryos. b – d Data are presented as Mean class="thinsp" contenteditable="false"> ± SD. b , c Kruskal–Wallis rank sum test followed by post hoc Steel-Dwass test, * p < 0.05, ** p < 0.01, and ns. d Unpaired two-tailed Mann–Whitney U test, ** p < 0.01.

Article Snippet: The synthesized cgRNA for trpc1 (5’-UCCAUGGUGGUGGAAUACUCguuuuagagcuaugcuguuuug-3’) and tracrRNA (5’-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3’), both of which act as the single-guide RNA, were obtained from Fasmac.

Techniques: Control, Injection, Irradiation, Knock-Out, Knockdown, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mechanochemical mechanism underlying intercellular Ca 2+ wave propagation and its crucial role in apoptotic cell extrusion

doi: 10.1038/s41467-025-65474-9

Figure Lengend Snippet: a – c Representative images of ACE in ( a ) DMSO-treated embryos (Control), ( b ) GsMTx4-treated embryos, and ( c ) trpc1 -/- embryos injected with piezo1- MO upon irradiation with a 15 nJ/pulse femtosecond laser through a 100×/1.25 NA objective lens. Apoptotic cells were identified by annexin V-FITC labeling. Red asterisks indicate the laser focal point. Dotted lines in x-z indicate the apical surface of epithelial cells. Scale bar: 20 µm. d Quantification of apical extrusion. Control: n = 12, GsMTx4: n = 11, and trpc1 -/- plus piezo1- MO: n = 9 embryos. two-sided Chi-squared, * p < 0.05, and ns.

Article Snippet: The synthesized cgRNA for trpc1 (5’-UCCAUGGUGGUGGAAUACUCguuuuagagcuaugcuguuuug-3’) and tracrRNA (5’-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3’), both of which act as the single-guide RNA, were obtained from Fasmac.

Techniques: Control, Injection, Irradiation, Labeling

Journal: Nature Communications

Article Title: Mechanochemical mechanism underlying intercellular Ca 2+ wave propagation and its crucial role in apoptotic cell extrusion

doi: 10.1038/s41467-025-65474-9

Figure Lengend Snippet: a Temporal dynamics of Ca²⁺ transients at representative junctions. To induce cell deformation without triggering apoptosis, a femtosecond laser at 10 nJ/pulse was focused on the site marked with a red asterisk through a 100×/1.25 NA objective lens. Control (DNSO, top panel), GsMTx4 (upper panel), CBX (lower panel), and trpc1 -/- plus piezo1 -MO (bottom panel). The right panels show kymographs of the white-boxed regions on the left. Scale bar: 20 µm. b The normalized GCaMP7 intensity of the apoptotic cell and adjacent surrounding cells, and displacement of the contact edge. Control (DMSO, top graph), GsMTx4 (upper graph), CBX (lower graph), and trpc1 -/- plus piezo1 -MO (bottom graph). Arrows indicate time points of laser irradiation. The shaded region represents the standard error of the mean. Control: n = 14 embryos, CBX: n = 8 embryos, GsMTx4: n = 8 embryos, and trpc1 -/- plus piezo1 -MO: n = 10 embryos.

Article Snippet: The synthesized cgRNA for trpc1 (5’-UCCAUGGUGGUGGAAUACUCguuuuagagcuaugcuguuuug-3’) and tracrRNA (5’-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3’), both of which act as the single-guide RNA, were obtained from Fasmac.

Techniques: Control, Irradiation

Journal: Nature Communications

Article Title: Mechanochemical mechanism underlying intercellular Ca 2+ wave propagation and its crucial role in apoptotic cell extrusion

doi: 10.1038/s41467-025-65474-9

Figure Lengend Snippet: a Schematic illustrations of force measurement using a femtosecond laser. The left panel shows cell extrusion induced by irradiation of the cell center with a femtosecond laser (15 nJ/pulse). The right panel shows that after Ca 2+ wave-mediated polarized movements are initiated (approximately 30 s after laser irradiation), a series of laser pulses (10 − 100 nJ/pulse through 100×/1.25 NA objective lens) are loaded every 1 s at the center of the extruding cell. b, c Force measurement during Ca 2+ wave-mediated polarized movements. b Red and yellow asterisks indicate the focal point of the laser to induce the extruding cell and loading of impulsive forces, respectively (representative of 38 independent samples). The white dotted line at 77 s shows the size of the extruding cell at 39 s. c Plot of the area of the extruding cell over time. An extruding cell was induced by laser irradiation at 0 s and gradually became smaller. By loading impulsive force, the size of the extruding cell slightly increased. After stopping the loading force, the extruding cell became smaller over time. Scale bar, 10 μm. d Pulse energy L dependence of threshold distance R to counter-balance the polarized movements. n = 38. e Force measurement during Ca 2+ wave-mediated polarized movements in embryos treated with DMSO (control, n = 34) GsMTx4 ( n = 25), XestoC ( n = 20), or 2-APB ( n = 31), and in trpc1 -/- embryos ( n = 21). One-way ANOVA followed by post hoc Tukey-Kramer test, ** p < 0.05, and ** p < 0.01. f Schematic illustration of Ca 2+ wave-mediated polarized movements during ACE proposed in this study. Cells surrounding the extruding apoptotic cell are deformed. This deformation triggers elevation of intracellular Ca 2+ via TRPC1 and CICR, resulting in cell shrinkage. Cell shrinkage induces deformation of surrounding cells located in Row 2. The increase of intracellular Ca 2+ triggers actin rearrangement in the cells, which leads to formation of c-lamellipodia. This chain reaction occurs in multiple rows of surrounding cells, leading to Ca 2+ wave-mediated polarized movements.

Article Snippet: The synthesized cgRNA for trpc1 (5’-UCCAUGGUGGUGGAAUACUCguuuuagagcuaugcuguuuug-3’) and tracrRNA (5’-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3’), both of which act as the single-guide RNA, were obtained from Fasmac.

Techniques: Irradiation, Control

Journal: Science (New York, N.Y.)

Article Title: Very Fast CRISPR on Demand

doi: 10.1126/science.aay8204

Figure Lengend Snippet: (A) Schematic of Cas9 activation by modulating base-pairing between the PAM distal region of cgRNA and genomic DNA. (B) Without light, Cas9/cgRNA bound to target DNA without cleavage, causing a clear band shift. Proteinase K degraded Cas9, causing target DNA to shift back to the original position. (C, D) Fast and efficient in vitro cleavage kinetics of Cas9 after light activation. (E) Indels detected by high-throughput sequencing of PCR-amplified genomic DNA extracted from cells without RNP, with RNP but no light, and with RNP 48 h after light activation. (F) DSBs detected by DSB-ddPCR of genomic DNA extracted from cells without RNP, with RNP but no light, and with RNP 30 s after light activation. (G-H) DSB % over time using our method (red) compared to either RNP electroporation (G, target sequence at ACTB) or a chemically inducible system (H, target sequence at MYC). (I) DSBs and Normalized Indels (Materials and Methods) at ACTB over time after Cas9 activation.

Article Snippet: Competing interests: The authors and Johns Hopkins University have filed a provisional patent application on the method of spatiotemporal control of Cas9 activities via cgRNA.

Techniques: Activation Assay, Electrophoretic Mobility Shift Assay, In Vitro, Next-Generation Sequencing, Amplification, Electroporation, Sequencing