Supplementary Figure 1 Custom-made perfusion lid for glass-bottom cell-culture dishes. (a) The perfusion lid has supported inlet and outlet holes located at the rim of the lid, thereby enabling liquid exchange on top of the plastic portion of the cell culture dish. This allows gentle injection and aspiration of liquid to and from the culture dish, without disturbing the neurons grown on the glass-bottom part of the dish. (b) The lid has 4 spacers to allow gas exchange between the culture dish and the microscope chamber. (c) A photograph of the perfusion lid with the glass-bottom cell culture dish. Note that the 2 mm aspiration tube going through the lid hole needs to be placed at a height where it is in close proximity to the plastic bottom of the culture dish (open arrow) to allow efficient aspiration of the buffer. The injection tubing can be placed further away from the dish bottom (arrow). (d) The perfusion chamber is secured tightly with clamps in the microscope chamber and (e) the injection and aspiration syringes are attached to the 2 mm tubes with blunt-end needles which can be changed when needed. Nature Protocols: doi:10.1038/nprot.2017.116
Transcript
Supplementary Figure 1
Custom-made perfusion lid for glass-bottom cell-culture dishes.
(a) The perfusion lid has supported inlet and outlet holes located at the rim of the lid, thereby enabling liquid exchange on top of the plastic portion of the cell culture dish. This allows gentle injection and aspiration of liquid to and from the culture dish, without disturbing the neurons grown on the glass-bottom part of the dish. (b) The lid has 4 spacers to allow gas exchange between the culture dish and the microscope chamber. (c) A photograph of the perfusion lid with the glass-bottom cell culture dish. Note that the 2 mm aspiration tube going through the lid hole needs to be placed at a height where it is in close proximity to the plastic bottom of the culture dish (open arrow) to allow efficient aspiration of the buffer. The injection tubing can be placed further away from the dish bottom (arrow). (d) The perfusion chamber is secured tightly with clamps in the microscope chamber and (e) the injection and aspiration syringes are attached to the 2 mm tubes with blunt-end needles which can be changed when needed.
Nature Protocols: doi:10.1038/nprot.2017.116
Supplementary Figure 2
Monitoring synaptic vesicle exocytic events with VAMP2-pHluorin to discriminate active presynapses from adjacent axons.
(a) A mosaic image of rat E18 hippocampal neurons cultured in a microfluidic device, and transfected with VAMP2-pHluorin on DIV14 and imaged on DIV16. Neurons are stimulated in high K
+ (both terminal and soma wells) and the fluorescence of VAMP2-pHluorin is
recorded in the nerve terminal chamber. (b and c) Quantification of VAMP2-pHluorin fluorescence of the hippocampal neuron shown in (a). The graphs show the measured fluorescence intensity for a representative (b) presynapse (boxed area in a) and (c) axonal segment (dashed box in a), in low K
+ (i.e. resting) and high K
+ (i.e. stimulated) conditions, demonstrating an increase in the average
fluorescence of the VAMP2-pHluorin (i.e. unquenching) in the presynapse upon release and exposure to the extracellular pH. Values presented in the graphs (b and c) show mean fluorescence after stimulation (high K
+) normalized to the mean fluorescence in the
resting neuron prior stimulation, i.e. in low K+. The values are shown as an example from n = 1 hippocampal neuron. Please see Box 1
for more details. Bar 10 µm. All the experiments were carried out in accordance with relevant institutional and governmental ethical guidelines and regulations (Animal Ethics Approval QBI/254/16/NHMRC).
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Supplementary Figure 3
Assessing synaptic identity, functionality and maturity.
(a) Cultured rat E18 hippocampal neurons are transfected with VAMP2-pHluorin (green) on DIV14, fixed on DIV16 and immunolabeled against endogenous Synapsin-1 (magenta). The boxed area in (a) is shown at higher magnification in (b) demonstrating the co-localization of the two synaptic markers in presynapses (arrowhead). (c) To assess synaptic functionality and correct localization of internalized nanobodies in recycling SVs, VAMP2-pHluorin-transfected neurons are subjected to sdTIM using HRP-tagged mCherry-GNT. After fixation and cytochemical staining, neurons are processed for electron microscopy. HRP-precipitate in SVs (open arrowheads) and unstained vesicles (arrowheads) are indicated. To assess the maturity of synapses in the terminal well of the microfluidic devices (d) VAMP2-pHluorin (green)-expressing neurons in the soma well are fixed and immunolabeled against endogenous PSD-95 (magenta) to identify dendrites in the terminal well. (e) Confocal image of the VAMP2-pHluorin-positive axon passing through a microfluidic channel and forming synaptic connections with the neurons cultured in the terminal well. (f) Higher magnification images from the boxed area in (e) show the punctate staining pattern of postsynaptic density (arrowhead) co-localizing with and adjacent to presynaptic VAMP2-pHluorin (open arrowhead). Bars are 5 µm (a and e), 0.1 µm (c), 100 µm (d) and 1 µm (b and f). See Box 2 for more details. All the experiments were carried out in accordance with relevant institutional and governmental ethical guidelines and regulations (Animal Ethics Approval QBI/254/16/NHMRC).
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Supplementary Figure 4
Single-molecule tracking with TrackMate.
Quantification of the SV mobility in presynapses (Ps) and axons (Ax) using the TrackMate plug-in for ImageJ. The graphs show (a) the frequency distribution of the average Log10 diffusion coefficient (D) and (b) MSD [µm
2]. The average numbers ±SEM are obtained from
presynapses (n = 6 presynapses from where 1,000 trajectories) and axonal segments (n = 12 axonal segments from where 3,600) of the same neuron presented in Fig. 4. All the experiments were carried out in accordance with relevant institutional and governmental ethical guidelines and regulations (Animal Ethics Approval QBI/254/16/NHMRC).
Nature Protocols: doi:10.1038/nprot.2017.116
Supplementary Figure 5
Estimation of localization precision using two methods.
(a-b) localization precision was estimated from the MSD curve. (a) A purely diffusive segment of the CTB trajectory shown in Fig. 6d was employed to estimate the localization precision (σ). (b) MSD [μm
2] curve of the trajectory segment shown in (a). The MSD curve
was fitted to the equation MSD(τ) = 4σ2 + 4Dτ to estimate the localization precision, where D is the diffusion coefficient and τ is the time
lag. The estimated σ (30 nm) was used to correct the diffusion coefficients estimated using HMM-Bayes analysis in Fig. 6. (c-d) Localization precision was estimated by fitting each fluorescent spot to a Gaussian distribution and localization precision was
approximated as s/ , where s is the standard deviation of the Gaussian function and N is the number of photons captured from a fluorescent spot. Distributions of localization precision of (c) Alexa647-CTB (~500 localizations) and (d) Atto565-NBs (>21,000 localizations) in hippocampal neurons. The average localization precision for Alexa647-CTB was 43 ± 9 nm and 32 ± 4 nm for Atto565-NBs. All the experiments were carried out in accordance with relevant institutional and governmental ethical guidelines and regulations (Animal Ethics Approval QBI/254/16/NHMRC).
Nature Protocols: doi:10.1038/nprot.2017.116
1
Supplementary Table1
sdTIM configuration Function /Localization References
sdTIM with labelled (e.g. Atto) hormones and neuropeptides
Regulate multiple brain and body functions (e.g. pain, social behaviour, etc.).
(10-12)
sdTIM with labelled (e.g. Atto) toxins and viruses
Cholera toxin Causative agent of cholera. Internalized by clathrin-independent endocytosis; transported from endosomes to Golgi and ER. Cytoplasmic penetration from ER.
(13-16)
Shiga toxin Causative agent of dysentery. Internalized by clathrin-independent endocytosis; transported from endosomes to Golgi and ER. Cytoplasmic penetration from ER.
(17, 18)
Botulinum toxin Activity-dependent retrograde trafficking from plasma membrane to endosomes and then to Golgi and ER.
(19, 20)
Anthrax toxin Clathrin and actin dependent endocytosis; cytoplasmic penetration from endosomes.
sdTIM of above-mentioned labelled (e.g. Atto647N) cargoes combined with sptPALM using genetically encoded markers tagged with photoconvertible fluorophore (mEos- or analogue)
Exocytosis
Sec4 Exocytic marker. (54)
Syntaxin1A
Plasma membrane resident soluble N-ethylmaleimide sensitive-factor attachment receptor proteins (SNARE) critical for the docking and priming of secretory vesicles from neurosecretory cells.
(55)
Munc18-1 Controls SNARE-dependent neuroexocytosis with syntaxin-1A.
(56)
Endocytosis
Clathrin, AP2, FCHO, etc. Components of clathrin-coated pits.
(57-60)
Caveolins, cavins Main components of caveolae.
(61-64)
Endophylin BAR domain protein. Induces membrane deformation and scission. Regulates ‘FEME’ endocytosis.
(65, 66)
Flotilins Flotilin -dependent endocytosis.
(67-69)
GRAF1 Regulates membrane remodelling in CLIC/GEEC endocytosis.
(70, 71)
Dynamin Maturation and scission of multiple endocytic invaginations.
(72-75)
Actin (LifeAct) Membrane deformation and (76, 77)
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3
scission, vesicle transport, cell motility and division.
RhoGTPases (e.g. Rac1, RhoA, Cdc42) and effectors (e.g. Pak, ROCK, Trio, etc.)
Actin remodelling during macropinocytosis, cell motility and division.
(78-80)
ARP2/3, Wasp, etc. Actin polymerization. (81, 82)
Cofilin, twinfilin, GMF, etc. Actin depolymerisation. (83-85)
Regulate non-vesicular lipid transport from ER to PM.
(115)
Rab5, EEA1, Early endosome formation and homotypic fusion.
(97-99)
APPLE1/2 Adaptor proteins localized on a subclass of signalling early endosomes.
(100)
Rab4, Rab11, Amphiphisin1/2, etc.
Formation and functioning of recycling endosomes.
(101, 102)
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