1. Doc2 Is a Ca2+ Sensor Required for Asynchronous Neurotransmitter Release 
Jun Yao, Jon D. Gaffaney, Sung E. Kwon, Edwin R. Chapman 
Cell 
Volume 147, Issue 3, Pages (October 2011)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
PS Is a Critical Doc2α and β Effector during Regulated Membrane Fusion
(A) In vitro membrane fusion assays were performed in the presence of 3 μM syt, Doc2α, or Doc2β. All three proteins accelerated fusion upon addition of 1 mM Ca2+ (arrow). No fusion was observed when SNARE proteins were omitted from the vesicles (ø).
(B) A Ca2+ titration for each protein was performed using 15% PS vesicles. Data were fitted with sigmoidal dose-response curves to determine the [Ca2+]1/2 values for fusion (inset).
(C) Syt or Doc2 titrations were carried out using vesicles with 0, 15 and 25% PS, and data were fit as in (B) to calculate EC50 values (0% PS: not applicable; 15% PS: syt, 0.5 μM; Doc2α, 2 μM; Doc2β, 0.9 μM; 25%: syt, 0.6 μM; Doc2α, 0.3 μM; Doc2β, 0.2 μM). Each point represents the mean ± SEM from three independent experiments.
Cell  , DOI: ( /j.cell )
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4. Figure 2
Doc2α and Doc2β Can Assemble Syntaxin and SNAP-25 into Functional SNARE Complexes
(A) The “traditional” fusion assay: vesicles harboring preassembled syntaxin⋅SNAP-25 heterodimers are incubated with syb-harboring vesicles and syt, Doc2α, or Doc2β.
(B) Fusion assays were carried out using “split t-SNAREs” in which syntaxin-alone was reconstituted into the t-SNARE vesicles and free SNAP-25 was subsequently added in a soluble form; under these conditions, fusion is not observed unless Ca2+ and syt—and, as tested here, Doc2—are added to drive folding of SNAP-25 onto syntaxin, resulting in fusion activity (Bhalla et al., 2006).
(C) Fusion assays using syt (left), Doc2α (center), or Doc2β (right) were carried out with preassembled t-SNARE vesicles or syntaxin vesicles plus free SNAP-25. In the presence of Ca2+, all three proteins were able to drive assembly of active t-SNARE heterodimers capable of fusing with syb vesicles.
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5. Figure 3
Doc2 Is Tuned to Respond to Changes in [Ca2+] with Markedly Slower Kinetics Than Syt
Stopped-flow rapid mixing experiments were performed to analyze the kinetics of syt, Doc2α, and Doc2β interactions with vesicles that harbor PS.
(A) Association kinetics were monitored via FRET between the endogenous aromatic residues of each protein and a dansyl-PE acceptor in the target vesicles.
(B) Representative association traces for each protein showing the first 500 ms (full traces for Doc2α and Doc2β are shown in Figure S2A).
(C) Each trace was fit with a double exponential function, and the rate of the fast component was plotted as a function of the liposome concentration. Each point represents the mean ± SEM from at least three independent experiments.
(D) The on rates, off rates, and dissociation constants were determined from the plots shown in (C).
(E) Disassembly of Ca2+⋅Doc2 or syt from liposomes upon rapid mixing with excess EGTA to chelate all Ca2+.
(F and G) (F) The first 200 ms of a representative syt disassembly trace and (G) the first second of representative Doc2α and Doc2β traces are shown. The rate of disassembly was determined for syt by fitting the data with a single exponential function; Doc2 data were fit with a double exponential.
(H) The data from at least five separate experiments were analyzed, and the mean ± SEM for each kinetic component and the τ values were determined.
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6. Figure 4
Doc2α KD Reduces Asynchronous SV Release in Syt I Knockout Hippocampal Neurons
(A and B) Immunoblots of Doc2α (A) or Doc2β (B) in cultured hippocampal neurons. Because Doc2β was not detected (B, left), the efficiency of Doc2β shRNA was examined in HEK293T cells transfected with a Doc2β-GFP construct (B, right; vertical line indicates lanes that were removed). Doc2α and Doc2β were reduced ≥ 79% by shRNA KD.
(C) Representative traces of evoked EPSCs recorded from syt I KO neurons (control) and lentivirus-infected KO neurons expressing Doc2α shRNA, Doc2β shRNA, or Doc2α shRNA plus Doc2β.
(D) Bar graph showing that the total charge transfer over 0.5 s is significantly reduced in the neurons expressing Doc2α shRNA (control, n = 34; shRNA, n = 34), but not Doc2β shRNA (n = 24) or Doc2α shRNA plus Doc2β (n = 23).
(E) Averaged total charge of 0.5 M sucrose-induced responses showing little difference between syt I KO neurons (n = 19) and KO neurons expressing Doc2α shRNA (n = 17).
(F) Representative EPSC traces from syt I KO neurons or KO neurons overexpressing WT Doc2α or Doc2α-CLM.
(G) Bar graph showing the total charge transfer during EPSCs recorded from syt I KO neurons (n = 33) and KO neurons overexpressing WT Doc2α (n = 16) or Doc2α-CLM (n = 40).
∗∗p < Error bars represent SEM.
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7. Figure 5
Doc2α KD Specifically Reduces the Asynchronous Phase of SV Release in WT Hippocampal Neurons
(A) Average traces of evoked EPSCs recorded from WT neurons and neurons preincubated with 25 μM AM-EGTA, as well as neurons expressing Doc2α shRNA alone or accompanied by expression of Doc2β.
(B) Bar graphs summarizing the amplitude of EPSCs recorded from WT neurons (n = 29), neurons treated with AM-EGTA (n = 67), and neurons expressing Doc2α shRNA alone (n = 42) or Doc2α shRNA plus Doc2β (n = 25).
(C) Average normalized cumulative EPSC charge transfer over 0.5 s demonstrating that neurons treated with AM-EGTA or expressing Doc2α shRNA alone significantly accelerate the decay kinetics of EPSCs, as compared to WT neurons and Doc2α KD neurons expressing Doc2β.
(D and E) Bar graphs summarizing the charge (D) and the time constant (E) of the slow phase of transmission.
(F and G) Bar graphs summarizing the charge (F) and the time constant (G) of the fast phase of transmission.
(H) Average traces of evoked EPSCs recorded from WT neurons (n = 18) and Doc2α KO hippocampal neurons (n = 23).
(I) Average normalized cumulative EPSC charge transfer over 0.5 s from WT neurons and Doc2α KO neurons.
(J and K) Bar graphs comparing the charge (J) and the time constant (K) of the slow phase of transmission between WT neurons (n = 18) and Doc2α KO neurons (n = 23).
(L and M) Bar graphs summarizing the charge (L) and the time constant (M) of the fast phase of transmission.
∗p < 0.05; ∗∗p < Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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8. Figure 6
Doc2α shRNA Induces Inverse Changes in the Synchronous and Asynchronous Components of Transmission during High-Frequency Train Stimulation
(A) Representative EPSC traces evoked by 40 action potentials at 20 Hz.
(B) Bar graph summarizing the RRP size measured by extrapolating the cumulative charge (top small panel) recorded from WT neurons (n = 33), neurons treated with AM-EGTA (n = 84), or neurons expressing Doc2α shRNA (n = 56).
(C) Bar graph showing that neurons treated with AM-EGTA or expressing Doc2α shRNA exhibited significant reductions in the fraction of total tonic charge transfer during the stimulus train. (Top) Diagram showing how the phasic and tonic charge were determined (Otsu et al., 2004).
(D) Average normalized cumulative tonic charge transfer over 400 ms demonstrating that AM-EGTA or Doc2α KD significantly decelerated the accumulation of tonic charge.
(E–G) Histograms summarizing the build-up of tonic current in WT neurons (E), neurons expressing Doc2α shRNA alone (F), and neurons treated with AM-EGTA (G). The data were fitted with Gaussian curves (red). Doc2α KD induced a left shift in the distribution, compared to WT neurons and Doc2β-rescued neurons. AM-EGTA neurons exhibited a disperse distribution and were not fitted.
(H) Typical traces of the first EPSCs during the train showing the extent of decay prior to the next stimulation. The amplitudes were normalized for comparison.
(I) Bar graph showing that both Doc2α KD and AM-EGTA treatment induce faster EPSC decay rates.
(J) Doc2α KD induced slower depression of EPSC amplitudes at the beginning of the train compared to control neurons; AM-EGTA treatment gave rise to the same decrease in depression as Doc2α KD during the late stage of the train.
∗∗p < Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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9. Figure 7
Doc2α KD Reduces Evoked Reverberatory Activity in High-Density Cultures of Hippocampal Neurons
(A) Sample image of a high-density hippocampal culture used to generate reverberatory activity. Scale bar, 100 μm.
(B) Representative traces of evoked reverberation recorded from WT neurons (control) and neurons expressing Doc2α shRNA.
(C and D) Histograms summarizing the number of reverberatory EPSCs that occurred within 2 s (C) and the duration of the reverberatory activity (D) in WT control (n = 22; left) and Doc2α KD neurons (n = 27; right).
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10. Figure S1
Ca2+ Promotes the Interaction of Doc2 and Syt with PS-Bearing Vesicles and Membrane-Embedded t-SNAREs, Related to Figure 1
(A) t-SNARE vesicles lacking PS were incubated with syt, Doc2α, or Doc2β in the presence of 0.2 mM EGTA or 1 mM Ca2+. Vesicles and bound protein co-floated through a density gradient and were collected. Samples were separated by SDS-PAGE and the gels stained with Coomassie blue.
(B) Since syntaxin and Doc2β co-migrate on SDS-PAGE, samples were also subjected to immunoblot analysis with a Doc2β antibody to confirm Doc2β binding. SNAP-25 served as a loading control.
(C) Representative gel of a co-sedimentation experiment. Protein-free liposomes harboring 25% PS were incubated with syt, Doc2α, or Doc2β in the presence of 0.2 mM EGTA or 1 mM Ca2+. Liposomes were sedimented by centrifugation and a fraction of each supernatant was collected and analyzed by SDS-PAGE and Coomassie blue staining; binding was monitored via depletion of protein from the supernatant.
(D) The fraction of bound protein was calculated and plotted as a function of [lipid].
(E–G) Doc2-regulated fusion is dependent on the formation of functional trans SNARE complexes. Fusion assays were performed in the presence and absence of syt (E) Doc2α (F) and Doc2β (G). Addition of either 10 μM cd-syb or the use of protein-free (PF) vesicles resulted in the complete loss of fusion activity.
Cell  , DOI: ( /j.cell )
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11. Figure S2
Kinetics of Syt and Doc2 Interactions with PS-Bearing Liposomes, Related to Figure 3
(A) Representative averaged traces (from ten trials in one experiment) for the assembly of Ca2+⋅protein⋅vesicle complexes on an expanded time scale. Liposomes harboring 5% Dansyl-PE and 25% PS plus 0.2 mM Ca2+ were rapidly mixed with an equal volume of 4 μM syt, Doc2α or Doc2β at 14°C. Final concentrations after mixing were: protein = 2 μM; liposome = 22 nM; Ca2+ = 0.1 mM.
(B and C) Disassembly of the Ca2+⋅protein⋅vesicle complexes as a function of temperature and %PS. Preassembled complexes were rapidly mixed with excess EGTA using a stopped-flow spectrometer; disassembly was monitored via loss of FRET between endogenous aromatic residues of Doc2α (B) or Doc2β (C) and dansyl-bearing liposomes. The rate of disassembly was determined at three different temperatures using vesicles containing either 15% (left column) or 25% PS (middle column). Right column, the τ values for disassembly were determined from two independent experiments and plotted as a function of temperature.
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12. Figure S3
Quantification of Doc2α KD and Lack of Effect on the Total Charge Transfer of Minis and Characterization of Two Doc2 Mutants, Related to Figure 4
(A) Representative immunoblot of Doc2α in cultured hippocampal neurons with and without shRNA mediated KD.
(B) Expression of Doc2α was reduced to ∼21% (20.8 ± 6.3%, n = 5) of wt levels by Doc2α shRNA.
(C) Representative traces of mEPSCs showing similar kinetics between syt I KO neurons and KO neurons with Doc2α KD.
(D) Quantitative analysis of total charge transfer during mEPSCs revealed that there was no difference between syt I KO neurons and KO neurons with Doc2α KD (control, n = 12; Doc2α KD, n = 10). (E) Typical EPSC traces recorded from syt I KO neurons overexpressing Doc2β or Doc2β-4A.
(F) Bar graph showing the total charge transferred during EPSCs recorded from syt I KO neurons overexpressing Doc2β (n = 33) or Doc2β-4A (n = 28).
(G) Immunocytochemistry, using an antibody specific for the luminal domain of syt I (604.1), revealed that TM-Doc2α, a chimeric protein consisting of the luminal, transmembrane, and linker domains of syt I and the C2AB domain of Doc2α, was localized to presynaptic boutons (and likely to SVs), which were labeled with a synapsin I antibody. Scale bar, 10 μm.
(H) Typical EPSC traces from syt I KO neurons, or KO neurons expressing TM-Doc2α (with and without AM-EGTA treatment).
(I) Bar graph showing the total charge transfer during EPSCs recorded from syt I KO neurons (n = 28), and KO neurons expressing TM-Doc2α (n = 30).
(J) Average normalized cumulative EPSC charge transfer over 500 ms showing similar kinetics between syt I KO neurons and KO neurons expressing TM-Doc2α. ∗p < 0.05, ∗∗p < Error bars represent SEM.
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13. Figure S4
Doc2α KD or AM-EGTA Reduces the Asynchronous Phase of SV Release in WT Hippocampal Neurons
(A and B) Representative EPSC traces of patch clamp recordings of syt I KO hippocampal neurons (A) and quantitative analysis of total charge transfer (B) confirmed that 25 μM AM-EGTA efficiently inhibits asynchronous SV release (control, n = 50; AM-EGTA, n = 36).
(C and D) Doc2α KD did not affect the kinetics of mEPSCs as revealed by averaged traces (C) and by the cumulative charge transfer (D) (wt, n = 12; Doc2α KD, n = 13).
(E) Histograms summarizing the time constant (τ) of the cumulative charge transfer for EPSCs, using a single exponential fitting method, in wt neurons, neurons treated with AM-EGTA, and neurons expressing Doc2α shRNA alone or Doc2α shRNA plus Doc2β. The data were fit with Gaussian curves (red). Both Doc2α KD and AM-EGTA treatment induced a shift to the left, as compared to wt neurons and Doc2α KD neurons rescued by Doc2β.
(F and G) Histograms summarizing the rise time (F) and decay time (G) of EPSCs. Both Doc2α KD and AM-EGTA treatment induced a shift to the left in the decay time, whereas the rise time was affected only by AM-EGTA.
(H) Bar graph showing that neurons treated with AM-EGTA, or expressing Doc2α shRNA, exhibited significant reductions in the τ for charge transfer during single action potential evoked release, as determined by single exponential fitting.
(I) Bar graph summarizing the rise time of EPSCs. Only AM-EGTA treated neurons exhibited a significant reduction as compared to wt neurons.
(J) Bar graph showing that neurons treated with AM-EGTA, or expressing Doc2α shRNA, exhibited significant reductions in the decay time of EPSCs. ∗p < 0.05, ∗∗p < Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Overexpression of Doc2 Specifically Enhances Asynchronous SV Release in WT Hippocampal Neurons, Related to Figure 5
(A) Immunoblots of neurons infected with lentivirus to overexpress Doc2α or Doc2β.
(B) Representative recordings of evoked EPSCs from wt hippocampal neurons or neurons overexpressing Doc2α or Doc2β.
(C) Average normalized charge transfer of EPSCs over 0.5 s demonstrating a slower charge accumulation in neurons overexpressing Doc2α or Doc2β, compared to wt neurons.
(D and E) Analysis of the charge of the fast (D) and slow (E) components of the EPSCs, demonstrating that Doc2α or β overexpression increase the amount of asynchronous but not synchronous release (control, n = 39; Doc2α, n = 43; Doc2β, n = 31), as compared to wt control.
(F and G) Neither Doc2α nor Doc2β altered the time constant of either phase of release.
(H) Averaged mEPSC traces from wt neurons and wt neurons overexpressing Doc2α or β.
(I) Average normalized cumulative charge transfer of mEPSCs over 0.5 s; no differences were observed between wt neurons and neurons overexpressing Doc2α or β (wt, n = 12; Doc2α, n = 11; Doc2β, n = 12). ∗p < Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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15. Figure S6
Doc2α KO Neurons Exhibit Inverse Changes in the Synchronous and Asynchronous Components of Transmission during High-Frequency Train Stimulation
(A) Representative traces of EPSCs evoked by 40 action potentials at 20 Hz.
(B) Bar graph showing that Doc2α KO neurons (n = 23) exhibited significant reductions in the fraction of total tonic charge during the 20 Hz train stimulation, as compared to wt neurons (n = 22).
(C) Doc2α KO neurons exhibited slower depression of EPSC amplitudes. ∗∗p < Error bars represent SEM.
Cell  , DOI: ( /j.cell )
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