mCLING reveals that stimulation-induced synaptic vesicle recycling occurs at the

As mentioned in the introduction, one of the most intriguing questions in the IHC field is the localization of membrane retrieval hotspots that could supply SV recycling. So far two possibilities have been derived from different technical approaches. On one hand, several EM studies have shown important processes of membrane recycling happening at the cell base upon stimulation, close to the synaptic active zones, suggesting a local recycling (Siegel and Brownell, 1986; Spicer et al., 1999, 2007; Lenzi et al., 2002; Kamin et al., 2014). On the other hand, studies using fluorescence imaging of FM 1-43 proposed a pathway starting with endocytosis at the apical pole of the cell, followed by membrane sorting in organelles at the cell top, like the Golgi apparatus or the ER, from where SVs are reformed and delivered to the active zones. (Griesinger et al., 2002, 2004, 2005). However, the already exposed concerns about the use of FM dyes in IHCs make this last model controversial. With the use of mCLING I expect to solve the long-standing question about the origin of recycling SVs in IHCs.

Another unanswered question in IHC’s physiology is the nature of the tubular structures often described by EM in the literature, and sometimes proposed as source of SV reformation (Siegel and Brownell, 1986; Spicer et al., 2007). Their identity has been difficult to define because, as explained at the beginning of section 3.3, constitutive membrane recycling and synaptic vesicle recycling occur in the same cellular volume in IHCs.

Therefore, mCLING is a potential tool to dissect the morphology and physiology of both constitutive and synaptic recycling, and to elucidate which of those are the tubules more related to.

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In a first approach to answer these questions, transversal melamine sections of mCLING-labeled IHCs were imaged to study the morphology and distribution of recycling organelles.

With the aim to discriminate between constitutive- and synaptic-related events, different labeling strategies were used:

- Resting condition: intended to evaluate only constitutive membrane recycling. OCs were incubated for 3 minutes at 37°C in HBSS without Ca²⁺ containing 1.7 µM mCLING. After incubation, OCs were briefly washed in mCLING-free buffer and directly fixed in 4% PFA and 0.2% glutaraldehyde. Embedding and sectioning were performed as described before.

- Stimulation condition: intended to evaluate constitutive membrane recycling and synaptic vesicle recycling. OCs were incubated for 2 minutes at 37°C in HBSS without Ca²⁺ containing 1.7 µM mCLING. OCs were immediately transferred to HBSS high K⁺ (65 mM) with 1.7 µM mCLING for 1 minute, to stimulate synaptic vesicle exocytosis.

After incubation OCs were briefly washed in mCLING-free HBSS without Ca²⁺ and directly fixed in 4% PFA and 0.2% glutaraldehyde. Embedding and sectioning were performed as described before.

- Recovery condition: intended to follow how the endocytosed structures containing mCLING are later processed by the different trafficking pathways. The same protocol used in the stimulation condition was applied but after high K⁺ and before fixation the OC was incubated for 5 minutes at 37°C in mCLING-free HBSS with Ca²⁺. This buffer has only 5 mM K⁺ and allows the cells to recover after strong SV release before they are fixed. Embedding and sectioning were also performed.

One of the first features I found when imaging melamine sections in high-resolution STED microscopy, was that the staining of the plasma membrane was not always uniform. It has been previously reported that during the fixation procedure some of the membrane phospholipids are solubilized by aldehyde fixatives, and removed by detergents when combined with immunostaining (Doggenweiler and Zambrano, 1981). Extracted phospholipids can leave behind pores that in sections look as discontinuities of the membrane. Such level of detail was not possible with conventional confocal microscopy.

Evidence of these pores was already shown in Figure 3.5A, right panel, where fixed cells were easily permeated by PI. Similarly, discontinuous membranes were also seen in fixed and permeabilized cultured hippocampal neurons (see section 3.4).

STED images from IHC transversal sections revealed abundant endocytosed structures in

83 both resting and stimulated conditions. This indicates that constitutive membrane traffic is relatively strong in IHCs (Figure 3.12A). The top and nuclear levels were dominated by large and elongated structures resembling tubules, with the presence of large and small round structures. In contrast, the cell base contained mainly small round vesicle-like structures and larger endosome-like ones of different sizes and shapes (from round to elongated).

Stimulated cells seemed to have more endocytosed structures at the cell base than resting ones (Figure 3.12A). In the recovery condition, the sizes of the tubular structures at the cell top and nucleus, as well as the organelles seen at the cell base were reduced, likely due to processes of budding and vesicle reformation. This is also supported by a more homogeneous size and distribution of labeled organelles. These observations go in line with previous results obtained in our laboratory using FM 1-43 photo-oxidation combined with EM to track recycling organelles in IHCs (Kamin et al., 2014), as well as with other EM studies (Siegel and Brownell, 1986; Lenzi et al., 1999, 2002; Kamin et al., 2014).

A MatLab script (written by Silvio O. Rizzoli) was used to quantify the percentage of area occupied by mCLING labeled organelles from the total area delimited by the plasma membrane in transversal sections. The analysis was divided in the aforementioned cellular levels (top, nuclear and basal) among the three different treatments. While endocytic uptake at the top and nuclear levels of the IHC seemed to be not affected by synaptic stimulation, it was greatly increased by more than two-fold at the basal level, when compared to the resting condition (Figure 3.12C). In the recovery condition the area occupied by mCLING-labeled organelles was reduced, probably due to two reasons: the breaking up of endosome-like structures and tubules into small vesicles that are more difficult to detect by the analysis, and the recycling of labeled organelles back to the plasma membrane.

Important aspects of IHC physiology can be derived from these results: first, even in resting conditions IHCs seem to have a very active membrane recycling activity, when compared, for example, with cultured COS7 cells incubated with mCLING for a longer period (5 minutes, Figure 3.4C and Figure 3.7B). Second, that synaptic vesicle recycling seems to occur only in the basal region of the IHCs, and not at the top and nuclear regions as it was suggested before (Griesinger et al., 2002, 2004, 2005). Third, it is likely that the abundant membrane uptake at the IHC top and nuclear levels represents constitutive trafficking.

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Figure 3.12 mCLING reveals recycling organelles in IHCs.

A. When incubated at 37°C, mCLING was found in organelles of different shapes and sizes in both resting (top panels) and stimulated cells (lower panels). While the top and nuclear levels were dominated by tubular and round structures, the basal level was populated mostly by round organelles including vesicle-like ones. Stimulation increased mCLING staining mostly at the base of the cell. Scale bars, 2 µm. B. Zoom-ups corresponding to the dashed line squares depicted in A.

The upper panel shows typical tubular-like structures found at the cell top and nuclear levels, while the lower panel shows the more homogeneous profile of the cell base. Scale bars, 500 nm.

C. Endocytosis levels were quantified as the area occupied by mCLING-labeled organelles.

Stimulation increases mCLING endocytosis only in the basal region of the cells. The bars show mean % normalized to the resting condition ± SEM, from 27, 22 and 57 cell top and nuclear regions (resting, stimulation and recovery, respectively), and from 25, 28 and 27 cell bases (resting, stimulation and recovery, respectively). n.s., no significant difference (t-test, P > 0.05).

***, significant difference (t-test, P ≤ 0.001).