Three-Dimensional Reconstruction of Inner Hair Cells Reveals the Vesicle Recycling Pathway

The analysis of typical electron micrographs from the different time intervals (zero calcium, high potassium stimulation, 5 minutes, 30 minutes) allow a rather vague interpretation of the morphology of the vesicle recycling process in IHCs. Thus, I reconstructed three-dimensional images of the different treated IHCs to better describe the morphological nature of the organelles that are involved in the vesicle retrieval process.

As a first step I cut serial cross-sections of 100 nm from the organ of Corti and imaged the IHCs using a transmission electron microscope. The individual micrographs of a single serial section were afterwards merged to obtain full image of the entire IHC. All merged serial sections were aligned using Photoshop. See Figure 3.26 for aligned serial sections of the

“zero calcium” (A) and “stimulated” (B) IHCs and Figure 3.27 for “5 minutes” (A) and “30 minutes” (B) IHCs. The shape of the IHCs is almost identical for all conditions (bottle-like structure). The cuticular plate is visible as a dense area at the apical tip of the IHCs (from where the stereocilia emerge). Additionally, in all sections mitochondria are visible as black spots (note that the photo-oxidized darkening of the mitochondria was not related to vesicle recycling (Grabenbauer et al., 2005)). The nuclei are visible in each single section in the middle of the IHCs.

Figure 3.26: Three-dimensional processing of IHC sections. Aligned IHC serial cross-sections build from merged individual electron micrographs (# indicate the section number).

Yellow arrowheads indicate the darkened mitochondria, cyan arrowheads the cuticular plate, and green arrowheads indicate the nucleus. (A) Zero calcium condition. (B) High potassium stimulation. Note that the serial sections of the “stimulated” preparation (B) show two adjoining hair cells (the nucleus of the left cell disappears with deeper cutting).

Figure 3.27: Three-dimensional processing of IHC sections. Aligned IHC serial cross-sections build from merged individual electron micrographs (# indicate the section number).

Yellow arrowheads indicate the darkened mitochondria, cyan arrowheads the cuticular plate, and green arrowheads indicate the nucleus. (A) Stimulated IHC after 5 minutes rest. (B) Stimulated IHC after 30 minutes rest.

In the further processing I used the serial sections to generate three-dimensional reconstructions of the individual IHCs. Cochlear IHCs of the mouse typically have a diameter of around 5-10 m and a length between approximately 30-40 m. These dimensions made it difficult to reconstruct entire IHCs and thus the reconstruction was limited to around 20 1 sections per condition (resulting already in approximately 2000 electron micrographs per IHC). With a section depth of 100 nm in total, more than 2 m along the longitudinal axis of

one hair cell was reconstructed (the true thickness of a single section was slightly bigger than 100 nm, as the preparations shrink by approximately 25% during electron microscopy processing (Gaffield et al., 2006)). For the three-dimensional reconstruction the plasma membrane, the nucleus, the cuticular plate, the active zones, and the labeled organelles were manually redrawn with Photoshop and saved as single images (see Methods for example images of redrawn structures). The IHCs were then reconstructed from these images assuming a thickness of 100 nm for each section, using routines written in Matlab.

Figure 3.28: Three-dimensional reconstruction of the FM1-43 labeled and photo-oxidized zero calcium IHC. The plasma membrane is represented in yellow, the cuticular plate in cyan, the nucleus in green, the labeled organelles in purple, and the active zone in red (red arrowhead indicates the location of the ribbon). The reconstruction is made of 21 serial cross-sections, each with a section depth of 100 nm.

The three-dimensional reconstructions of the IHCs under zero calcium and stimulation conditions (Figure 3.28 and Figure 3.29, respectively) displayed an involvement of large

cisternae (and not tubules) in the membrane recycling process. Their primary location was essentially at the baso-lateral site. Interestingly, as already observed from single electron micrographs they disappeared in the IHCs with the resting periods (compare Figure 3.30 (5 minutes) and Figure 3.31 (30 minutes) with Figure 3.28). The reconstructed stimulation IHC, as observed before, showed a more numerous appearance of round organelles at the basal part, compared to the zero calcium IHC. All reconstructed IHCs showed labeled organelles at the cuticular plate (apex). Nevertheless, in all reconstructions the major membrane labeling was located below the nucleus, suggesting that the apical membrane retrieval might be just a minor recycling route. Interestingly, by comparing the control (zero calcium) and the 5 minutes conditions by eye the baso-lateral sites were characterized in the former by cisternae and in the latter by high frequencies of small labeled organelles. This indicates that a morphological change of the organelles has occurred. The large elongated cisternae likely disintegrated into round vesicle-like organelles. A morphological analysis of the reconstructions is presented below.

Figure 3.29: Three-dimensional reconstruction of the FM1-43 labeled and photo-oxidized high potassium stimulation IHC. The plasma membrane is represented in yellow, the cuticular plate in cyan, the nucleus in green, the labeled organelles in purple, and the active

zone in red (red arrowhead indicates the location of the ribbon). The reconstruction is made of 19 serial cross-sections, each with a section depth of 100 nm. Note that here two neighboring IHCs are partially reconstructed.

Figure 3.30: Three-dimensional reconstruction of the FM1-43 labeled and photo-oxidized high potassium stimulation and 5 minutes rest IHC. The plasma membrane is represented in yellow, the cuticular plate in cyan, the nucleus in green, the labeled organelles in purple, and the active zone in red (red arrowhead indicates the location of the ribbon). The reconstruction is made of 21 serial cross-sections, each with a section depth of 100 nm.

Figure 3.31: Three-dimensional reconstruction of the FM1-43 labeled and photo-oxidized high potassium stimulation and 30 minutes rest IHC. The plasma membrane is represented in yellow, the cuticular plate in cyan, the nucleus in green, the labeled organelles in purple, and the active zone in red (red arrowhead indicates the location of the ribbon). The reconstruction is made of 21 serial cross-sections, each with a section depth of 100 nm.