Role of formins and mDia1/3 in SV endocytosis.
(A) Maxima of background-corrected Syph-pHluorin fluorescence traces (surface normalized) for neurons treated with 0.1 % DMSO (1.7 ± 0.1) or JLY cocktail (1.8 ± 0.2) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. N = 4 independent experiments from nDMSO = 23 videos; nJLY = 36 videos.
(B) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalization) for neurons treated with 0.1 % DMSO or JLY cocktail (1.0 ± 0.1) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values for DMSO were set to 1. N = 4 independent experiments from nDMSO = 23 videos; nJLY = 29 videos.
(C) Averaged normalized Syph-pH fluorescence traces from transfected hippocampal neurons treated with 0.1% DMSO, JY or JL combinations (containing 8 µM Jasplakinolide, 5 µM Latrunculin A and 10 µM Y-27632) stimulated with 200 APs (40 Hz, 5s). Data shown represent the mean ± SEM. N = 4 independent experiments from nDMSO = 24 videos; nJY = 19 videos; nJL = 21 videos.
(D) Endocytic decay constants of Syph-pHluorin traces in C: τDMSO = 19.0 ± 1.9 s; τJY = 28.5 ± 1.6 s; τJL = 18.2 ± 1.6 s; pDMSO vs JY < 0.01, one-way ANOVA with Tukey’s post-test. Data shown represent mean ± SEM.
(E) Averaged normalized Syph-pH fluorescence traces from transfected hippocampal neurons treated with 0.1% DMSO or 10 µM Y-27632 following 200 AP (40 Hz, 5s) stimulation. Data shown represent the mean ± SEM. N = 4 independent experiments from nDMSO = 25 videos; nY = 18 videos.
(F) Endocytic decay constants of Syph-pHluorin traces in C: τDMSO = 17.0 ± 2.2 s; τY = 20.2 ± 4.3 s. Data shown represent mean ± SEM.
(G) Maxima of background-corrected Syph-pHluorin fluorescence traces (surface normalized) for neurons transfected with shCTR (1.8 ± 0.1) or shmDia1 (1.8 ± 0.1) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. N = 9 independent experiments from nshCTR = 49 videos; nshmDia1 = 42 videos.
(H) Analysis of knockdown efficiency of lentiviral particles carrying shRNA against no mammalian target (shCTR) or mDia1 and mDia3 (shmDia1+3) in mouse hippocampal cultures harvested 12 days after transduction. Protein abundance of mDia1, mDia3 and Tubulin were immunoblotted with specific antibodies.
(I) Averaged normalized vGLUT1-pHluorin fluorescence traces from stimulated (40 APs; 20 Hz, 2 s) hippocampal neurons transduced with lentiviruses encoding shCTR, shmDia1 or both shmDia1 and shmDia3 combined (shmDia1+3). Data represent mean ± SEM. N = 4 independent experiments from nshCTR = 17 videos, nshmDia1 = 19 videos, nshmDia1+3 = 18 videos. The corresponding endocytic decay constants are shown in Figure 1G.
(J) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons transduced with shCTR (1.9 ± 0.1) or shmDia1+3 (1.9 ± 0.1) in response to 40 AP stimulation (20 Hz, 2 s). Data represent mean ± SEM. N = 22 independent experiments from nshCTR = 105 videos and nshmDia1+3 = 128 videos.(K) Averaged normalized vGLUT1-pHluorin fluorescence traces for neurons transduced with shCTR, shmDia1 or shmDia1+3 in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N = 4 independent experiments from nshCTR = 12 videos; nshmDia1 = 15 videos; nshmDia1+3 = 18 videos. Corresponding endocytic decay constants are shown in Figure 1H.
(L) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons transduced with shCTR (2.8 ± 0.2), shmDia1 (2.6 ± 0.2), or shmDia1+3 (2.4 ± 0.1) in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N = 4 independent experiments from nshCTR = 12 videos; nshmDia1 = 15 videos and nshmDia1+3 = 18 videos.
(M) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalized) for neurons transduced with shCTR or shmDia1+3 (1.0 ± 0.2) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values for shCTR were set to 1. N = 11 independent experiments from nshCTR = 45 videos and nshmDia1+3 = 42 videos.
(N) Schematic representation of the activation of mDia1. Binding of RhoA-GTP to the RBD domain (green) or application of mDia1 activator (IMM) competes with the intramolecular interaction of the N-terminal DID (yellow) with the C-terminal DAD (red) domain (see Figure 3A for domain structure). The release of autoinhibition leads to dimerization of mDia formins in solution.
(O) Maxima of background-corrected vGLUT1-pHluorin fluorescence traces (surface normalized) for neurons treated with 0.1 % DMSO (1.7 ± 0.1) or mDia activator (IMM; 1.6 ± 0.1) in response to 80 AP stimulation (40 Hz, 2 s). Data represent mean ± SEM. N = 3 independent experiments from nDMSO = 18 videos; nIMM = 16 videos.