Figure 4

STIM1/TRPC1-dependent SOCE mediates the spontaneous and netrin-1-potentiated filopodial Ca²⁺entries. (A) Left panel; a Lck-GCaMP3 fluorescent Ca²⁺ image of a Xenopus spinal growth cone showing three ROIs (F1, F2 and F3) encompassing the filopodia used to measure fluorescent intensities over time (right panels). Scale bar, 10 μm. The incidence (B) and the frequency (C) of the spontaneous filopodial Ca²⁺ transients were significantly attenuated by XSTIM1-DN (n = 30) or XTRPC1-MO (n = 30), when compared to the control (n=34; *P < 0.001 and **p < 0.005, Student’s t-test). Values represent mean ± s.e.m. (D) A Lck-GCaMP3 fluorescent Ca²⁺ image of a growth cone showing three ROIs (left panel) and their representative traces of Ca²⁺ signals in three filopodia (F1, F2, F3) before and after bath application of netrin-1 (10 ng/ml) (right panel) in the presence of Sp-cAMP (25 μM). Scale bar, 10 μm. ( E-F ) Netrin-1 potentiated the incidence (E) and the frequency (F) of filopodial Ca²⁺ transients in spinal growth cones (control; n = 14) and this potentiation was abolished by XSTIM1-DN (n = 8) and XTRPC1-MO (n = 10). **P < 0.005 and ***p < 0.05 (Student’s t-test). Values represent mean ± s.e.m. (G) Filopodia tips are the major site of initiation of filopodial Ca²⁺ entry as revealed by kymographs of Ca²⁺ signals in filopodia using Lck-GCaMP3 in modified Ringers saline (MR; n = 67), netrin-1 exposure (n = 27) and Ca²⁺ re-addition after depletion (SOCE; n = 43). The y axis represents the path distance along the filopodia divided into 10 portions and the x axis represents time. The arrows denote the tip and base of filopodia.