Low-Mobility of Fused Synaptic Vesicles

After synaptic vesicle exocytosis the vesicle material is located on the plasma membrane at the AZ. The vesicle recycling models hypothesize that the vesicle material is mobile in the membrane in order to move to the endocytic periactive zone. As the fused vesicle material recycles rapidly after exocytosis (Westphal et al., 2008) it is difficult to explore its mobility on the membrane.

Several methods can be devised to investigate fused vesicle mobility. The mobility under natural conditions can be recorded in absence of divalent ions in the extracellular buffer. The lack of divalent ions blocks endocytosis and also calcium-triggered exocytosis. The material resides on the plasma membrane at an equilibrium state (Figure 3.12 A; (Zefirov et al., 2006b)). Nevertheless, the amount of fused vesicle material can be increased in the absence of divalent ions by stimulating exocytosis with caffeine (Zefirov et al., 2006a) or with the black widow spider venom (BWSV, (Ceccarelli and Hurlbut, 1980; Henkel and Betz, 1995).

Treatment of neurons with caffeine in the absence of divalent ions efficiently causes exocytosis by releasing a minor concentration of calcium from intracellular stores, such as the endoplasmatic reticulum. This concentration is sufficient to trigger exocytosis and results in the addition of a small fraction of vesicle material to the one on the plasma membrane under equilibrium conditions. Quite contrary to caffeine, the BWSV treatment causes massive exocytosis events with almost the entire synaptic vesicle pool being forced to fuse with the plasma membrane. Endocytosis can at the same time be blocked by the lack of calcium in the buffer (Ceccarelli and Hurlbut, 1980). The synaptic vesicle materials are then retained in the plasma membrane of a swollen synapse (Figure 3.12 A).

These three conditions (control (no divalents), caffeine and BWSV) were used on hippocampal cultured neurons to investigate the mobility of the fused vesicle pool on the plasma membrane.

Figure 3.12: Blocking endocytosis allows the investigation of fused synaptic vesicles. (A) Schematic representation of the experimental setup. Endocytosis can be inhibited by decreasing the concentrations of divalent ions in the extracellular medium (no divalents, left) to specifically investigate the mobility of the surface pool of synaptotagmin at equilibrium.

The fraction of this pool can be increased by stimulation while simultaneously blocking endocytosis using caffeine (not shown) or BWSV (right). Please consider that BWSV results in an excessive increase in fused vesicle material contrary to caffeine induced stimulation.

(B) Hippocampal neurons were treated with BWSV, caffeine or divalent-free Tyrode with a subsequent staining of the surface pool of synaptotagmin with directly fluorescence-labeled Oyster-550 monoclonal mouse anti-synaptotagmin antibodies (Syt, green) on ice. After different incubation times at RT the preparations were fixed and the surface retained monoclonal antibodies were immunostained (without permeabilizaton) using Cy5-tagged secondary antibodies (Sytsurface, red). Scale bar: 5 µm. (C) The relative amount of antibody on the surface was determined and expressed as the fraction of the control condition (fixed immediately after labeling). The bars show the mean SE from three independent experiments for each condition. The dotted line indicates the amount of synaptotagmin left exposed under natural conditions after only 2 min of incubation (Westphal et al., 2008). Note that for all time points considered, all three conditions allow a large fraction of synaptotagmin molecules to remain on the surface instead of being endocytosed.

Before the actual STED imaging was performed the reliability of the three different protocols for blocking endocytosis was tested. The fraction of remaining surface-exposed synaptotagmin was analyzed after different time intervals. To test for this the neurons were first treated using the respective protocols (BWSV: 15 minutes incubation at 37 °C; caffeine:

5 minutes incubation at RT; control: 5 minutes incubation at RT). Afterwards, the fused

synaptic vesicle material was live-labeled on ice with directly fluorescence-labeled Oyster-550 antibodies against synaptotagmin. The neurons were then switched to RT and fixed at different time points (fixation on ice after 0 (control), 5, 10, and 20 minutes), followed by immunostaining (without permeabilization) with Cy5-tagged secondary antibodies for the specific detection of the surface retained synaptotagmin protein. Images of the total labeled synaptotagmin pool (green, Oyster-550) and the surface pool of synaptotagmin (red, Cy5) were acquired (Figure 3.12 B). The amount of fused synaptotagmin left on the plasma membrane after the particular time points was then quantified. The expected block of endocytosis was fulfilled in all three conditions used. Even after 20 minutes approximately two times more material remained on the surface in all three treatments when compared to the amount left on the plasma membrane after 2 minutes under natural conditions (shown as the dotted black line, see (Westphal et al., 2008)). The results revealed the strength of the endocytosis block, with the most efficient inhibition observed for the caffeine treatment.

The mobility of the fused synaptic vesicle material was then investigated using live STED imaging. The neurons were treated using these protocols and the surface-exposed synaptotagmin proteins were live-labeled with directly fluorescence-labeled anti-synaptotagmin antibodies (604.2 tagged with Atto647N obtained from Synaptic Systems, Göttingen). The mobility of the surface pool is shown in Figure 3.13 A (see Movie A6). Note the swollen synapse in the BWSV treated sample, indicating the massive amount of fused synaptic vesicle material (upper row of Figure 3.13 A).

Figure 3.13: Mobility of fused synaptic vesicles. (A) Hippocampal cultures were treated with BWSV, caffeine or divalent-free Tyrode and the surface pool of synaptotagmin was live-labeled. The panels show three typical movie frames (frames #1-3 in each row) from BWSV (upper panel), caffeine (middle panel) or divalent-free (lower panel) preparations (see Movie A6). The gray panels show the sum images (500 frames) of the movies from the respective row. Each sum image has characteristical “hot spots”, indicating the preferred location of fused vesicle material. Scale bar: 250 nm. (B) The graph shows histograms of the median trace speed of the fused vesicle material (BWSV: black; caffeine: grey; no divalents:

punctuated black). Note that under natural conditions (no divalents) the material on the membrane is limited in mobility. The mobility increased by adding more fused vesicle material to the plasma membrane via stimulation with BWSV or caffeine. The histograms were computed from 2500–3000 values.

Tracking of the fused vesicle material resulted in relatively low speed values under control conditions (no divalents, Figure 3.13 B). Note that the mobility was almost identical to the

preparations showed faster vesicle movements on the plasma membrane compared to the control condition. This indicates that the surface pool of synaptotagmin at normal conditions is somehow hindered in its movements. However, the increased fraction of fused vesicles by BWSV or caffeine resulted in a more unrestricted movement, indicating that these added vesicles escaped the mechanism that is responsible for the low-mobility state under normal conditions (control).