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. 2016 Dec 7;92(5):1020-1035.
doi: 10.1016/j.neuron.2016.10.014. Epub 2016 Nov 10.

Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses

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Free PMC article

Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses

Xin-Sheng Wu et al. Neuron. .
Free PMC article

Abstract

Mechanical force is needed to mediate endocytosis. Whether actin, the most abundant force-generating molecule, is essential for endocytosis is highly controversial in mammalian cells, particularly synapses, likely due to the use of actin blockers, the efficiency and specificity of which are often unclear in the studied cell. Here we addressed this issue using a knockout approach combined with measurements of membrane capacitance and fission pore conductance, imaging of vesicular protein endocytosis, and electron microscopy. We found that two actin isoforms, β- and γ-actin, are crucial for slow, rapid, bulk, and overshoot endocytosis at large calyx-type synapses, and for slow endocytosis and bulk endocytosis at small hippocampal synapses. Polymerized actin provides mechanical force to form endocytic pits. Actin also facilitates replenishment of the readily releasable vesicle pool, likely via endocytic clearance of active zones. We conclude that polymerized actin provides mechanical force essential for all kinetically distinguishable forms of endocytosis at synapses.

Figures

Figure 1
Figure 1. Actb−/− and Actg1−/− calyces
(A) Antibody staining of β-actin, γ-actin, and vGluT1 in P9 control (Ctrl), Actb−/−, and Actg1−/− calyces (images overlay in the right). (B) β- and γ-actin staining intensity (mean + s.e.m., AU, arbitrary unit) in P7-10 Ctrl (54 calyces, 3 mice), Actb−/− (56 calyces, 3 mice), and Actg1−/− calyces (53 calyces, 3 mice). **: p<0.01 (t test, compared to Ctrl).
Figure 2
Figure 2. β- or γ-actin knockout inhibits slow endocytosis at calyces
(A–C) Sampled ICa and Cm (A), mean Cm traces (mean + s.e.m., B), and Ratedecay, ΔCm and QICa (mean + s.e.m., C) induced by depol20ms (arrow) from Ctrl (13 calyces, 13 mice, black), Actb−/− (12 calyces, 8 mice, red) and Actg1−/− (12 calyces, 9 mice, blue) calyces from P7-10 mice at 22–24°C. s.e.m. is plotted every 1 s. **, p<0.01 (t test, applies to other plots). The first 0.5 s Cm trace after depol20ms, which may contain Cm artifacts, was not shown (applies to all Cm trace in Fig. 2). (D–F) Similar to A-C, except that the stimulus was 20 APe at 100 Hz (P7-10, 22–24°C). Control, n = 7 calyces, 7 mice; Actb−/−, n = 6 calyces, 6 mice; Actg1−/−, n = 6 calyces, 6 mice. (G) Cm traces and Ratedecay (mean + s.e.m.) induced by depol20ms from Ctrl (8 calyces, 8 mice) and Actb−/− (8 calyces, 8 mice) calyces at 34–37°C (P7-10). **, p<0.01 (t test). (H) Cm traces and Ratedecay (mean + s.e.m.) induced by depol20ms from Ctrl (10 calyces, 9 mice) and Actb−/− (9 calyces, 9 mice) calyces in P13-14 mice (22–24°C). **, p<0.01 (t test).
Figure 3
Figure 3. β- or γ-actin knockout inhibits rapid endocytosis at calyces
(A–H) Similar arrangements as Fig. 2A–H, respectively, except that the stimulus was depol20msX10 (A–C, G–H) or 200 APe at 100 Hz (D–F). A–C: Ctrl, 13 calyces, 13 mice; Actb−/−, 12 calyces, 8 mice; Actg1−/−, 12 calyces, 9 mice (P7-10, 22–24°C). D–F: Ctrl, 7 calyces, 6 mice; Actb−/−, 6 calyces, 5 mice; Actg1−/−, 6 calyces, 5 mice (P7-10, 22–24°C). G: Ctrl, 8 calyces, 8 mice; Actb−/−, 8 calyces, 8 mice (P7-10, 34–37°C). H: Ctrl, 10 calyces, 9 mice; Actb−/−, 9 calyces, 9 mice (P13-14, 22–24°C). *, p<0.05; **, p<0.01 (t test).
Figure 4
Figure 4. β- or γ-actin knockout inhibits bulk endocytosis and an endocytic step before fission pore closure
(A–B) Sampled Cm (A) and DCS frequency (B, mean ± s.e.m) induced by depol50msX10 with 5.5 mM calcium in the bath from Ctrl (n = 24 calyces), Actb−/− (n = 29 calyces), and Actg1−/− (n = 18 calyces) calyces. The arrow points to a DCS in Ctrl (A, left). DCS frequency, binned every 20 s, is plotted versus time before and after depol50msX10 (time 0). (C) Sampled Cm with a DCS and an accompanying Gp shown enlarged in the inset from a Ctrl, Actb−/−, and Actg1−/− calyx. Ctrl trace is the same as the Ctrl trace in A. The stimulation was depol50msX10 with 5.5 mM calcium in the bath (also applies to D). (D) The mean (+s.e.m.) rate of Gp change and initial Gp during DCSs in control (43 DCSs), Actb−/− (15 DCSs), and Actg1−/− (9 DCSs) calyces. No statistical difference was observed (p > 0.14, t test).
Figure 5
Figure 5. β-actin or γ-actin knockout inhibits endocytosis overshoot and the RRP replenishment
(A) Averaged Cm changes (mean + s.e.m.) induced by depol50msX10 (arrow) from control (24 calyces), Actb−/− (29 calyces) and Actg1−/− (18 calyces) mice (bath: 5.5 mM calcium). Dotted line: baseline. (B–C) The endocytosis overshoot amplitude (mean + s.e.m.) measured at 40 s after depol50msX10 (B) and the Ratedecay after depol50msX10 (C) in control (n = 24), Actb−/− (n = 29) and Actg1−/− (n = 18) calyces (bath: 5.5 mM calcium). A positive value means an overshoot. **: p <0.01, t test. (D) Sampled Cm induced by depol20msX10 (each arrow: 1 depol20ms) from Ctrl, Actb−/− and Actg1−/− calyces. The Cm jump induced by the first depol20ms was normalized for comparison. Bath: 2 mM calcium (applies to panel D–G) (E) ΔCm (left) and the accumulated ΔCm (ΣΔCm, right) induced by each of the 10 depol20ms during depol20msX10 in Ctrl (13 calyces), Actb−/− (12 calyces) and Actg1−/− (12 calyces) calyces. Data (mean ± s.e.m.) are normalized to the ΔCm induced by the 1st depol20ms. (F) Sampled Cm traces induced by a pair of depol20ms at an interval of 200 ms in a Ctrl and a Actb−/− calyx. (G) Left: the ratio between the 2nd and the 1st ΔCm (ΔCm2/ΔCm1) during a pair of depol20ms plotted versus paired-pulse interval (each data point: 5–7 calyces). Right: same as in left, but plotting the interval between 0–1 s. *: p < 0.05; **: p < 0.01 (t test).
Figure 6
Figure 6. β- or γ-actin knockout inhibits endocytosis at hippocampal synapses
(A) Western blot of β-actin, AP-2, Clathrin heavy chain (CLT), dynamin (DMN) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, loading control) from ActbLoxP/LoxP hippocampal cultures transfected with nothing (Ctrl) or with lentivirus containing a Cre enzyme (Actb−/−). (B) Similar to A, except from Actg1LoxP/LoxP cultures. (C) Upper: western blot of β-actin, γ-actin and GAPDH from Cre-ERTM;ActbLoxP/LoxP culture at 0, 2 and 4 days (d) after addition of 4-OH-tamoxifen (1 μM). Lower: β- (left) and γ-actin (right) western blot intensity from Cre-ERTM;ActbLoxP/LoxP culture at 0, 2 and 4 days after addition of 4-OH-tamoxifen (mean + s.e.m., normalized to day 0, n = 4). ***, p < 0.001 (ANOVA). (D) SypH and mCherry images of a neuron transfected with SypH and a plasmid containing Cre-mCherry. mCherry is localized to nucleus (superimposed image), due to a nuclear localization sequence tagged at the Cre N-terminal. The box region is enlarged (right) to indicate a place for SypH imaging. (E) FSypH (mean + s.e.m.) induced by Train10s (left, n = 8 experiments) or Train2s (n = 5) in control boutons, and FSypH induced by Train10s in Actb−/− boutons (ActbLoxP/LoxP boutons transfected with SypH and a Cre plasmid, n = 11) or in Actg1−/− boutons (Actg1LoxP/LoxP boutons transfected with SypH and a Cre plasmid, n = 10). FSypH is normalized to baseline, s.e.m. is plotted every 10 s, temperature was 22–24°C (applies to Figs. 6–8 if not mentioned otherwise). (F) Traces in E (same color coding) scaled to the same amplitude and superimposed. Train2s was aligned to the end of Train10s. (G) Ratedecay and ΔF (mean + s.e.m.) induced by Train10s and Train2s in control boutons (Train10s, n = 8; Train2s, n = 5) and by Train10s in Actb−/− (n = 11) and Actg1−/− boutons (n = 10 experiments). ΔF was normalized to baseline F (ΔF/F, applies to all panels in Figs. 6–7). **: p < 0.01, t test (compared to Train10s data in Ctrl). (H) Applying MES solution (pH:5.5, bars) quenched FSypH (mean + s.e.m.) to a similar level (dotted line) before and after Train10s in Actb−/− boutons (n = 6 experiments). ΔS represents pre-existing SypH molecules at the plasma membrane that can be quenched. (I) FSypH traces, Ratedecay and ΔF (mean + s.e.m.) induced by Train10s in Ctrl (n = 4, black) or Actb−/− boutons (n = 5 experiments, red) at 34–37°C. Mean FSypH traces were also scaled and superimposed (right). *: p < 0.05; **: p < 0.01, t test. (J) FSypH traces, Ratedecay and ΔF (mean + s.e.m.) induced by a 10 s train at 80 Hz at 22–24°C in Ctrl (n = 4, black) or Actb−/− boutons (n = 5 experiments, red). Mean FSypH traces were also scaled and superimposed (right). **: p < 0.01, t test.
Figure 7
Figure 7. Actin polymerization is needed for endocytosis
(A–B) FSypH traces (A), Ratedecay and ΔF (B) induced by Train10s (bar) in Ctrl hippocampal boutons (n = 8 experiments), in Actb−/− boutons transfected with β-actin (Actb−/−/β rescue; transfection of β-actin with SypH and a Cre-mCherry plasmid in ActbLoxP/LoxP boutons; n = 6), and in Actb−/− boutons transfected with γ-actin (Actb−/−/γ rescue, n = 6). Data are expressed as mean + s.e.m. (C–D) Similar to A–B, but for Actg1−/− boutons transfected with γ-actin (Actg1−/−/γ rescue, n = 8) or β-actin (Actg1−/−/β rescue, n = 8). (E–F) Similar to panel A–B, but for Ctrl boutons (n = 8), Actb−/− boutons (n = 11), and Actb−/− boutons transfected with β-actin(G13R) [Actb−/−/G13R rescue: transfection of β-actin(G13R) with SypH and a Cre-mCherry plasmid in ActbLoxP/LoxP hippocampal boutons; n = 8]. **: p < 0.01, t test.
Figure 8
Figure 8. Ultrastructural changes in TM4d-Actb−/− hippocampal boutons
(A) EM images of wild-type and TM4d-Actb−/− hippocampal boutons fixed at rest (R) and at 0, 3 and 10 min after the end of 1.5 min 90 mM KCl application. For R, HRP was included for 1.5 min; for KCl application, HRP was included only during KCl application (see labels). (B–C) The number of HRP(+) vesicles, HRP(−) vesicles, and their sum (B), and the bulk endosome area (C) per μm2 of synaptic cross section are plotted versus the time before (R) and at 0 (K+), 3, and 10 min after the end of KCl application in control and TM4d-Actb−/− hippocampal cultures (mean + s.e.m., each group was from 40–100 synaptic profiles). ***, p < 0.001; **, p < 0.01; *, p < 0.05 (ANOVA, also applies to E). (D) EM images of membrane pits with various shapes obtained during or after KCl application from either control or TM4d-Actb−/− culture. h and b refer to pit height and base length. (E) The number of pits before (R) and after KCl application in control and TM4d-Actb−/− synapses (mean + s.e.m.).

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