Endocytic processes reported by mCLING are dynamin- and clathrin-dependent87

In Figure 3.11 I showed that incubation of IHCs with mCLING at low temperatures resulted in inhibition of its uptake. This important outcome suggests the potential of mCLING as marker for endocytic activity levels. Hence, I used mCLING to study the effects of inhibiting two important molecules previously involved in conventional endocytosis (see Introduction), and also in SV recycling at conventional synapses (Brodin et al., 2000):

clathrin and dynamin.

In order to inhibit clathrin-mediated endocytosis I used pitstop 2, a small molecule (473 Da) that binds to the terminal domain of the clathrin heavy chain and hinders its association with other proteins like amphiphysin, AP180 and Synpatojanin1, resulting in inhibition of clathrin coat formation (von Kleist and Haucke, 2011; von Kleist et al., 2011). To inhibit dynamin activity I used dynasore, another small molecule (322 Da) that permeates the plasma membrane and acts as a noncompetitive inhibitor of the GTPase activity of dynamin (Macia et al., 2006; Newton et al., 2006). Dynasore has been used to inhibit synaptic vesicle recycling in conventional synapses (Chung et al., 2010; Hoopmann et al., 2010; Watanabe et al., 2013) and in cells containing ribbon-type synapses like photoreceptors (Van Hook and Thoreson, 2012; Wahl et al., 2013) and IHCs (Duncker et al., 2013).

To study the effects of clathrin and dynamin inhibition in stimulated IHCs, OCs were treated during 25 minutes with either pitstop 2 (30 µM) or dynasore (100 µM) in HBSS without Ca²⁺. Afterwards an stimulation protocol was applied as described before: 2 minutes in HBSS without Ca²⁺ with 1.7 µM mCLING, in presence of the inhibitor (same concentration), followed by 1 minute HBSS high K⁺ (65 mM) with mCLING and the inhibitor (same concentrations). OCs were washed, fixed and immunostained for the standard vesicular marker of IHCs, the Vesicular Glutamate Transporter 3 (VGLUT3). OCs were embedded in melamine and cut into 200 nm sections. Control cells were incubated with mCLING using the same protocol and buffers, but in absence of the inhibitors.

STED images of treated cells show that pitstop 2 reduced the amount of endocytosed organelles throughout the IHC. The tubular structures normally seen at the cell top and

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nuclear levels were reduced in number. At the cell base very few structures were visible (Figure 3.14A). The same quantification analysis used in Figure 3.12C was applied to determine the area occupied by mCLING-labeled structures, reporting a reduction of endocytosis down to ∼33% at the cell top/nuclear levels when compared to untreated stimulated cells. Endocytosis levels at the cell base went down to ∼7% (Figure 3.14B).

Figure 3.14 Effects of clathrin and dynamin inhibition visualized by mCLING labeling in IHCs.

A. IHCs were incubated with the clathrin inhibitor pitstop 2 (30 µM) for 25 minutes, and were then stimulated using 65 mM KCl, for 1 minute in buffer containing mCLING. Endocytosed organelles were less abundant than in untreated cells (see Figure 3.12 for reference). Scale bars, 2 µm. B. The amount of mCLING endocytosis at the top/nuclear and basal levels of pitstop 2 treated cells was compared to the corresponding values in control cells. Error bars represent mean percentage ± SEM from 63 cell top/nuclear areas and 52 cell bases treated with pitstop 2.

C. IHCs were incubated with the dynamin inhibitor dynasore (100 µM) for 25 minutes and stimulated as described for pitstop 2. Here endocytosis was also reduced at all cell levels. Scale bars, 2 µm. D. Quantification of mCLING endocytosis in dynasore treated cells compared to control cells. Error bars represent mean percentage ± SEM from 54 cell top/nuclear areas and 100 cell bases treated with dynasore. In general, clathrin and dynamin inhibition had stronger effects at the cell base of IHCs. E. Example of a dynasore-treated cell presenting mCLING-labeled structures accumulated at the cell base plasma membrane (white arrowhead). Right panels show a zoom in of the depicted area (white dashed-line square). Scale bars, 2 μm and 500 nm, respectively.

Dynasore had the same effects on endocytosis as pitstop 2, with reduced amount of mCLING-labeled structures at all cellular levels (Figure 3.14C). Endocytosis levels were

89 reduced to 59% at the cell top/nuclear levels, and to 15% at the cell base (Figure 3.14D).

Interestingly, while most of the dynasore-treated IHCs had reduced amount of organelles, a few cells presented large clumps of mCLING-labeled structures retained at the plasma membrane of the cell bottom (Figure 3.14E).

These results show that inhibition of both, clathrin and dynamin, had stronger effects at the cell basal level than at the top and nuclear levels. Considering the data previously presented, I could suggest that these two proteins have an important role in SV recycling. The molecular mechanisms supporting the remaining endocytosis at the upper levels remain unclear, as they seem to be more clathrin- and dynamin-independent.

3.3.8 Impairment of synaptic vesicle exocytosis reduces mCLING-reported endocytosis

The high rates of SV release found in IHCs have to be compensated by equally efficient mechanisms of membrane retrieval. The equilibrium between both processes is important to guarantee availability of synaptic vesicles and their proteins for future rounds of exocytosis, as well as to keep a constant surface area of the cell. One interesting question is if the impairment of SV release has also an impact in the levels of endocytosis. To answer this question I applied mCLING to OCs dissected from animals lacking the protein otoferlin, involved in SV exocytosis and priming (Roux et al., 2006; Pangršič et al., 2010). Otoferlin knockout mice (Otof^-/-) have pronounced hearing impairment due to a drastic reduction of exocytosis levels (Roux et al., 2006).

OCs from Otof^-/- mice were incubated with mCLING in the same stimulation conditions described before (section 3.3.5). OCs were subsequently fixed, immunostained for VGLUT3, embedded in melamine and cut into 200 nm sections. STED images show an important reduction of endocytosed organelles, in a similar way to the inhibition of clathrin and dynamin (Figure 3.15A). When compared to wild type animals, endocytosis was reduced to

∼32% at the cells top/nuclear levels, and to ∼8% at the cell base (Figure 3.15B).

These data are consistent with the model of basal synaptic recycling, suggesting that an impairment in SV release entails reduction in compensatory endocytic processes. It is not clear, however, how the reduction on SV recycling would affect endocytosis at the top and nuclear levels of the IHCs. Overall, these experiments demonstrate that mCLING indeed reports endocytic membrane uptake and that it can be easily used for its quantification.

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Figure 3.15 Impaired SV exocytosis by deletion of the protein otoferlin is accompanied by reduced levels of endocytosis in IHCs.

A. IHCs from otoferlin-deficient animals (Otof^-/-), in which exocytosis is completely abolished, were stimulated in the presence of mCLING. Endocytosis was reduced, especially at the base of the cell. Scale bars, 2 µm. B. The amount of endocytosis was compared to that in wildtype cells at the top/nuclear levels and at the basal level of Otof^-/-. Error bars represent mean percentage ± SEM from 28 cell top/nuclear areas and 26 cell bases. Values are presented as percentage of the wildtype condition.