3.3 mCLING elucidates membrane trafficking pathways in IHCs
3.3.3 mCLING labeling and sample processing for the study of recycling organelles in
The images shown in Figure 3.9 strongly suggest that with mCLING a novel endocytosis marker that fulfills the requirement of IHCs has been found. However, achieving a detailed
77 study on membrane recycling organelles in IHCs meets other limitations:
- The organ of Corti is a thick and complex tissue, where IHCs are surrounded by cells of other types (e.g. pillar cells and supporting cells), and covered on the apical side by the tectorial membrane. Although confocal microscopy has been useful to study the general labeling pattern of IHCs, endocytosis processes at smaller scales would be difficult to see.
- The accumulation of mCLING at the plasma membrane gives a strong fluorescence signal that could mask the weaker signal coming from small endocytosed organelles, even in confocal imaging.
- Electron microscopy studies have associated several types of organelles to synaptic vesicle recycling in IHCs: synaptic vesicles of 31 nm average diameter, larger clathrin-coated vesicles of 54 nm average diameter, and large cisternae of around 100 to 200 nm that resemble bulk endocytosis (Lenzi et al., 2002; Kamin et al., 2014;
Neef et al., 2014). Additionally, other structures that are presumably involved in constitutive membrane recycling include tubular structures with profiles of a few tens of nanometers distributed at the cell top and nuclear levels, as well as vesicles endocytosed right below the cuticular plate with sizes below 100 nm (Kachar et al., 1997; Spicer et al., 1999; Kamin et al., 2014). The small size of these structures, many times far below the Abbe’s diffraction barrier of light (200-300 nm), requires the implementation of a high-resolution fluorescence microscopy method.
These three difficulties were addressed by the implementation of an adapted protocol for sample preparation and imaging, described in the Methods section (2.2.5.1 and 2.2.5.2). A way to simplify the study of the organ of Corti and circumvent the first and second problems described above was to embed the mCLING-labeled organ of Corti in a water-based resin called melamine. When prepared, melamine resin is liquid and transparent, which is convenient for sample embedding and deep tissue penetration. After heating it up in steps of increasing temperature, melamine hardens and can be cut into sections as thin as 20 to 200 nm with a conventional ultramicrotome. These sections offer a clearer view of single organelles and bring the advantage of avoiding scattering background fluorescence from other planes of the sample, or bleaching fluorophores in the areas adjacent to the focal plane. Hence, different volumes of the cells can be imaged independently with increased detail. Additionally, melamine is non-fluorescent and due to its hydrophilic properties does not require sample dehydration, therefore offering good ultrastructure preservation (Punge
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et al., 2008; Punge, 2009).
For studying organelles with sizes below the sub-diffraction limit, presented above as the third limitation, I relied on our laboratory expertise implementing high-resolution stimulated emission depletion (STED) microscopy to different biological preparations. Our laboratory has successfully used STED to study endosomal and synaptic vesicle trafficking in cultured cells and neurons (Barysch et al., 2009; Hoopmann et al., 2010; Opazo et al., 2010).
As explained in the Introduction (section 1.4), in this technique the sample is illuminated with an excitation beam and a doughnut-shaped depletion beam spatially overlapped. As a result, in the doughnut center, where only the excitation light is present, photons will be collected as in conventional fluorescence microscopy. However, in the doughnut border, where both beams coincide, fluorophores will be excited and then stimulated by the depletion beam to emit photons at longer wavelengths than usual, far from the fluorescence detection window (Klar et al., 2000). In this way, the effective excitation volume is modulated to achieve resolutions 16-fold higher in the planar axis than its confocal counterparts (Hoopmann et al., 2010). Moreover, STED microscopy is convenient over other high-resolution light microscopy techniques, because it instantly delivers sub-diffraction images without relying on further computational processing (Klar et al., 2000; Hell, 2007).
One limitation of commercial STED setups, like the one I used for this study, is that resolution improvement is only achieved in the X-Y plane, while in the axial direction resolution is similar to that of conventional light microscopy (500-600 nm). Fortunately, this drawback is compensated for by the sectioning of the melamine embedded samples.
Altogether, while STED microscopy provides lateral high-resolved images, melamine sectioning offers increased axial resolution, which can actually be modulated by the cutting thickness (See Figure 2.2).
Since one of the main objectives of this study is to characterize synaptic vesicle recycling in IHCs, an adequate stimulation protocol was necessary. Previous studies have used K+ solutions at concentrations ranging from 30 to 80 mM with stimulation periods between 10 and 30 minutes. We estimated that such incubation times are typically long and not comparable with the physiological situation. In a previous electron microscopy study we found that incubation of the OC in a 65 mM K+ solution for 1 minute, was enough to trigger important membrane recycling processes in IHCs (Kamin et al., 2014). With the present study I would like to not only confirm those results by high-resolution fluorescence microscopy, but also extend them with information on the molecular/organellar identity of
79 the recycling structures.
I proceeded incubating OCs with 1.7 µM mCLING for 1 minute. OCs were then fixed, embedded in melamine, and cut into 200 nm sections. When these sections were studied under epifluorescence microscopy, I found that mCLING penetration was poor, sometimes getting trapped in extracellular structures like the tectorial membrane (Figure 3.10A), or reaching only the membrane surrounding the hair bundle at the cell top, or the cell base with its neighboring afferents (Figure 3.10B). Extending the incubation period to 2-3 minutes ensured good mCLING penetration and endocytic uptake, as seen in the following results sections. When stimulation was required, high K+ was applied only in the last minute.
Figure 3.10 A 1-minute incubation period is not long enough for mCLING to penetrate into OCs and label IHCs homogeneously.
Organs of Corti were incubated with 1.7 µM mCLING solution for 1 minute, fixed with 4% PFA and 0.2% glutaraldehyde, embedded in melamine resin and cut into 200 nm thick sections.
Sections were imaged in epifluorescence microscopy. mCLING is shown in red. An autofluorescence picture (FITC channel in green) is presented as aid to determine the IHCs outline. A. pictures an example in which mCLING is retained by the tectorial membrane (TM), reaching only the hair bundle at the IHC apical pole (white asterisk). At the cell base mCLING labeled some of the synaptic terminals (white arrowheads), confirming that it can also diffuse from the OC bottom. B. shows a case in which mCLING had better penetration into the OC but could not be taken up by endocytosis, evidenced by the absence of labeled organelles inside the IHC. These pictures also prove that even when mCLING reaches the hair bundle, it does not permeate the MET channels, since no apical staining is present.