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Science 316 (5824): 570-574

Copyright © 2007 by the American Association for the Advancement of Science

A Selective Activity-Dependent Requirement for Dynamin 1 in Synaptic Vesicle Endocytosis

Shawn M. Ferguson1,2,3, Gabor Brasnjo5*, Mitsuko Hayashi1,2,3*, Markus Wölfel3, Chiara Collesi1,2,3,7, Silvia Giovedi1,2,3, Andrea Raimondi1,2,3, Liang-Wei Gong1,2,3, Pablo Ariel5,6, Summer Paradise1,2,3, Eileen O'Toole8, Richard Flavell1,4, Ottavio Cremona7, Gero Miesenböck3, Timothy A. Ryan5, and Pietro De Camilli1,2,3{dagger}

1 Howard Hughes Medical Institute, Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA.
2 Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510, USA.
3 Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
4 Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
5 Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
6 David Rockefeller Graduate Program, The Rockefeller University, New York, NY 10021, USA.
7 IFOM, the FIRC Institute for Molecular Oncology Foundation, and Università Vita—Salute San Raffaele, Milano, Italy.
8 Boulder Laboratory for 3D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.


Figure 1 Fig. 1.. Dynamin 1–KO mice appear normal at birth. (A) HT and KO pups several hours after birth. Arrows highlight less milk in the stomach of the KO pup. (B) Immunoblot analysis of cell lysates from primary cortical neuron cultures (15 to 21 DIV) with dynamin isoform-specific antibodies and a pandynamin antibody. Clathrin LC, clathrin light chain. [View Larger Version of this Image (52K GIF file)]
 

Figure 2 Fig. 2.. Synaptic transmission in primary cultures of dynamin 1–KO cortical neurons. Cumulative probability histograms of peak mEPSC (A) and mIPSC (B) amplitudes from WT (black lines) and KO cultures (red lines). The mean mEPSC amplitude was increased from 17.0 ± 1.8 pA in WT neurons to 24.8 ± 2.5 pA in the dynamin 1–KO neurons (t test, P = 0.011, n = 2495 and 2435 respectively). A similar increase in amplitude was observed for mIPSCs (mean for WT = 17.3 ± 0.8 pA, n = 2369 versus 22.2 ± 1.0 pA, n = 2731, t test, P = 0.0006). (C) Modest reduction in single evoked (2-ms depolarization to +30 mV) EPSC amplitudes [WT n = 45, KO n = 40, P = 0.0967 (t test)] and significant reduction in single IPSC amplitudes in KO neuron pairs [*P <0.001 (t test, n = 39, 38 for WT, KO)]. (D) Examples of evoked IPSCs for WT and KO respectively. Shown are the 1st, 3rd, and 7th IPSC responses to stimulation at 10 Hz. Presynaptic stimulation is indicated as membrane voltage (gray dotted lines). (E) IPSC depression curves for control (black) and KO (red) during a sustained 10-Hz stimulation. Averaged data points represent 16 (control) and 7 (KO) cell pairs, binned into nonoverlapping groups of 10 responses. (F) Slower recovery of IPSCs from depression induced by 1000 action potentials at 10 Hz (shown in E) as assessed by 0.1-Hz stimulation. Averages represent unbinned data from 12 control and 6 KO cell pairs. The recovery (mean of the last five points) was markedly slower in the KO (t test, P = 0.0024). [View Larger Version of this Image (30K GIF file)]
 

Figure 3 Fig. 3.. Ultrastructural defects in dynamin 1–KO synapses of cultured neurons. (A) WT synapse. (B and C) KO synapses revealing an abundance of clathrin-coated profiles (arrows highlight stalks connecting clathrin-coated buds), an increase in the average synaptic vesicle size, and the presence (C) of several abnormally large vesicles. (D) A KO synapse with a massive accumulation of interconnected clathrin-coated buds (arrowheads) and only a small cluster (arrow) of heterogeneously sized synaptic vesicles. An asterisk indicates an evagination of an adjacent cell into this nerve terminal. (Inset) A plasma membrane connected network of clathrin-coated pits observed in a serial section from this same synapse (~200 nm away). (E) Accessibility of clathrin-coated profiles (arrowheads) in KO neurons to cholera toxin-HRP (10 µg/ml for 5 min on ice) supports their connection to the plasma membrane (the arrow indicates the location of the synaptic vesicle cluster within this synapse). (F) Partial reconstruction of a dynamin 1–KO synapse from electron tomography data shows three branched tubular networks (pale green, blue, and yellow) capped by clathrin-coated pits (white arrows) that are connected to the plasma membrane (green) in close proximity to two synaptic vesicle clusters (SVs, blue). (G) Quantification of synaptic vesicle external diameter (black, WT; red, KO; 10-nm bins). Vesicles exceeding 80 nm in diameter were 5.4 times as abundant in KO as in WT synapses (87 vesicles in KO versus 16 vesicles in WT) but were excluded from the analysis presented in (G) as their identification as synaptic vesicles remained questionable. (H) Histogram of clathrin-coated profile (CCP) counts from 75 WT and 87 KO synapses. WT = 0.2 ± 0.05, KO = 4.7 ± 0.89 CCPs/synapse (means ± SEM, P < 0.0001, t test). Data derived from three independent experiments. Horizontal black line is the mean. Scale bars, 200 nm. [View Larger Version of this Image (152K GIF file)]
 

Figure 4 Fig. 4.. Activity-dependent synaptic vesicle recycling defects in dynamin 1–KO synapses. (A) Immunofluorescence for clathrin light chain and dynamin 3 reveals a predominantly diffuse distribution in cultured WT neurons, but a punctate and overlapping distribution in KO neurons (see also fig. S4). Treatment with TTX (1 µM, 16 to 24 hours) caused clathrin and dynamin 3 in the KO neurons to redistribute to a diffuse localization resembling the localization of these proteins in untreated WT neurons (scale bar, 10 µm). (B) Morphometric analysis of EM images demonstrating that the accumulation of clathrin-coated profiles in KO synapses was reversed following TTX treatment (1 µM, 16 to 24 hours). (C to I) EM analysis of synapses from cortical cultures of WT and KO brains incubated with the extracellular tracer HRP (10 mg/ml) in control Tyrode's buffer (90 s), following a 90-s stimulation with 90 mM KCl and then a further 10-min recovery period in Tyrode's buffer. (C) Quantification of changes in total synaptic vesicle number and in the number of vesicles positive for the extracellular tracer HRP. (D to I) Representative examples of HRP uptake by WT and KO synapses under the conditions described above. At rest, HRP-labeled clathrin-coated buds emerging from the labeled plasma membrane invagination are visible in the KO synapse (G). Following stimulation, the WT nerve terminal (E) contains labeled and unlabeled vesicles, whereas in the KO synapse (H) synaptic vesicles are nearly depleted (long arrow) and labeled clathrin-coated buds (short arrows) are visible. After recovery, synaptic vesicles, including labeled vesicles, are present in both genotypes (F and I). It is expected that, at this concentration, only a fraction of the endocytic vesicles should take up HRP. EM scale bar, 200 nm. [View Larger Version of this Image (124K GIF file)]
 

Figure 5 Fig. 5.. Frequency dependent impairment of synaptic vesicle endocytosis in dynamin 1–KO neurons (A) Representative traces from WT (left panel) and KO (right panel) synapto-pHluorin expressing neurons stimulated in the presence (blue circles) or absence (black squares) of bafilomycin (Baf). A 10-Hz field stimulation began at t = 0 and ended after 30 s (300 action potentials, no Baf) or 90 s (900 action potentials, Baf). (B) Brief application of extracellular, membrane impermeant acid rapidly quenches all surface synapto-pHluorin in the prestimulus period (KO neuron). Following a 30-s stimulus (end marked by arrow), the fluorescence is quenched to the same level as the prestimulus period. The average degree of quenching poststimulus was 94.0 ± 1.4% in WT (n = 5) and 94.6 ± 1.0% in KO (n = 8). (C) The pooled average kinetics of exocytosis (exo = {Delta}FBaf) from WT (blue) and KO (green) neurons after 900 action potentials (10 Hz in presence of bafilomycin) and the pooled average kinetics of endocytosis (endo = {Delta}FBaf{Delta}Fno Baf) from WT (red) and KO (cyan) neurons after 300 action potentials at 10 Hz (stimulus ends at first arrow). The dashed line indicates the extent of exocytosis at the 30-s time point where endocytosis to exocytosis ratios (endo/exo) are calculated. Error bars are shown at two time points on the endocytosis curves (n = 9 for both WT and KO). (D) The average endo/exo ratio after 300 action potentials at 10 Hz as determined by using either synapto-pHluorin (spH) or synaptotagmin1 (syt1)-pHluorin (gray bars). Rescue refers to dynamin 1–KO cells that were cotransfected with synapto-pHluorin and dynamin 1, dynamin 2, or dynamin 3. (E) Endo/exo ratios following 300 action potentials at different frequencies. (F) Mean Endo/exo ratios after 300 action potentials (10 Hz) in 0.75 mM [Ca2+]. The numbers shown in parentheses in (D, E, and F) represent the number of independent experiments and error bars in this figure show ± SEM. [View Larger Version of this Image (40K GIF file)]
 


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