![Actomyosin-II–dependent axon beading suppresses the long-range spreading of the [Ca2+]axonalong the stressed axon. (A) Summation of [Ca2+]axon intensities over the entire stress-induced beading process, featuring representative control and PNB (+PNB; 50 μΜ) treated axons. Bracketed regions are shown magnified in the lower boxes. The changes in fluorescence intensity (ΔF/F0) reflecting the kinetics of [Ca2+]axon fluctuations are color-coded. Bar = 20 μm. (B) Kymographs of magnified axons in A, with bursts of [Ca2+]axon wave indicated by red arrows on the right. [Ca2+]axon hot spots are indicated with asterisks. X-axis bar = 5 μm, y-axis bar = 200 s. (C and D) Quantification of the ΔF/F0 changes reflecting the kinetics of [Ca2+]axon fluctuations (C) in the total axonal area (N = 26, 42) and (D) in the beading regions (N = 28, 30). (E) Quantification of the Ir of D (N = 27, 30). (F) Quantification of the time required to decay to half-maximal ΔF/F0 using the data set in D (N = 28, 30). (G) The relative burst frequency of [Ca2+]axon in control (Ctrl) and PNB-treated axons (N = 28, 47). (H) Representative tiled live-cell images showing the [Ca2+]axon in the non-stressed (Soma) and stressed (Flux) regions. Yellow-boxed regions are magnified in the lower panels, with red arrows indicating the flux direction. [Ca2+]axon intensity is color-coded. Bar = 40 μm (top) and 20 μm (bottom). (I) Comparison of [Ca2+]axon in the non-stressed soma chamber (N = 33, 30). (J) In control or Blebbistatin (BLB) treated neurons, the SCG10 intensity in the stressed axonal regions follows the stress induced by the indicated flow rates. Bar = 150 μm. (K and L) Quantification of (K) SCG10 and (L) Cleaved Caspase 3. For SCG10: control, N = 80*, 105*, 71, 63; +BLB, N = 90, 82, 84; subsets of data marked with asterisks were used in Fig. 4, K and L, of Pan et al. [2022]). For Cleaved Caspase 3: control, N = 119*, 131*, 125, 105; +BLB, N = 84, 109, 114; subsets of data marked with asterisks were used in Fig. 4, I and J; of Pan et al. [2022]). Data represent mean ± SEM; in C, D, F, G, K, and L, unpaired two-tailed Student’s t test; in E, Mann-Whitney test; in I, paired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001; * marks the t test results with the 0 μl/min control dataset; # marks the t test results between control and BLB-treated neurons of the same flow rate.](https://cdn.rupress.org/rup/content_public/journal/jcb/223/8/10.1083_jcb.202206046/2/jcb_202206046_fig7.png?Expires=1771543610&Signature=y~94OJydRp0rxFlmNVF3l7GJVIdHKAyIuAGS-9LQL7sMjZoDrUXbO2MTI0MwEHPJ9Dz7bpWJmfVsX2h7TV4j~UnCOhCjA3-tpcQ~XKRI1WuxWOhV5CVp9l~2BHCDJSDSpPawBQrh-mxh~UPq7qjZdNT2MDET1ADuQ6zN2oqnICsAXNCE9j5oEk3Qth26kCykQBCrugODRMd-zd0Pmsp~odVQ0Yu4lO95NIpq6UHSWJeXpmAn~Wn9D-sB9WpsIX-G8n7ECNR8rmMgV6VPEjAcs0ZdoqGUYCW94jH68xZk3R80Q6YcG~0JbYgjwNEB3SZPSBUiaVzQYfFPCUj0x8fcAg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Actomyosin-II–dependent axon beading suppresses the long-range spreading of the [Ca 2+ ] axon along the stressed axon. (A) Summation of [Ca2+]axon intensities over the entire stress-induced beading process, featuring representative control and PNB (+PNB; 50 μΜ) treated axons. Bracketed regions are shown magnified in the lower boxes. The changes in fluorescence intensity (ΔF/F0) reflecting the kinetics of [Ca2+]axon fluctuations are color-coded. Bar = 20 μm. (B) Kymographs of magnified axons in A, with bursts of [Ca2+]axon wave indicated by red arrows on the right. [Ca2+]axon hot spots are indicated with asterisks. X-axis bar = 5 μm, y-axis bar = 200 s. (C and D) Quantification of the ΔF/F0 changes reflecting the kinetics of [Ca2+]axon fluctuations (C) in the total axonal area (N = 26, 42) and (D) in the beading regions (N = 28, 30). (E) Quantification of the Ir of D (N = 27, 30). (F) Quantification of the time required to decay to half-maximal ΔF/F0 using the data set in D (N = 28, 30). (G) The relative burst frequency of [Ca2+]axon in control (Ctrl) and PNB-treated axons (N = 28, 47). (H) Representative tiled live-cell images showing the [Ca2+]axon in the non-stressed (Soma) and stressed (Flux) regions. Yellow-boxed regions are magnified in the lower panels, with red arrows indicating the flux direction. [Ca2+]axon intensity is color-coded. Bar = 40 μm (top) and 20 μm (bottom). (I) Comparison of [Ca2+]axon in the non-stressed soma chamber (N = 33, 30). (J) In control or Blebbistatin (BLB) treated neurons, the SCG10 intensity in the stressed axonal regions follows the stress induced by the indicated flow rates. Bar = 150 μm. (K and L) Quantification of (K) SCG10 and (L) Cleaved Caspase 3. For SCG10: control, N = 80*, 105*, 71, 63; +BLB, N = 90, 82, 84; subsets of data marked with asterisks were used in Fig. 4, K and L, of Pan et al. [2022]). For Cleaved Caspase 3: control, N = 119*, 131*, 125, 105; +BLB, N = 84, 109, 114; subsets of data marked with asterisks were used in Fig. 4, I and J; of Pan et al. [2022]). Data represent mean ± SEM; in C, D, F, G, K, and L, unpaired two-tailed Student’s t test; in E, Mann-Whitney test; in I, paired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001; * marks the t test results with the 0 μl/min control dataset; # marks the t test results between control and BLB-treated neurons of the same flow rate.
