Morphological Analysis of Recycling Organelles in Inner Hair Cells

The three-dimensional reconstructions revealed already much information about the morphology of the vesicle recycling pathway in IHCs. However, for a more detailed view on the morphological changes of the endocytosed organelles I performed a morphological analysis of the recycling organelles. The analysis should address the questions (1) of what nature are the recycling organelles and (2) where are the different organelles located? Thus, I used different tools to analyze the distribution and the size of the organelles.

In a first step the positive photo-oxidized organelles were counted to describe the average density. The IHC at 5 minutes showed approximately twice as many labeled organelles (on average 4 organelles per m²) compared to the zero calcium and the stimulated IHCs (2.1

and 1.6 organelles per m², respectively; Figure 3.32). However, after 30 minutes rest fewer labeled organelles were present compared to the 5 minutes time point (on average around 2.6 organelles per m²). One can infer from the density increase after 5 minutes and its decrease after 30 minutes that the membrane was retrieved via large infoldings (large membranes exhibit lower organelle densities), from which then small vesicles bud off, which results in the higher density of labeled organelles at 5 minutes. These vesicles could then participate again in neurotransmitter release and thus lose the FM dye, which is shown by the decrease of labeled organelles after 30 minutes.

Figure 3.32: Density of labeled organelles. Shown are the numbers of organelles present per m² for the four three-dimensional reconstructed IHCs.

Furthermore, I analyzed the size and shape of the labeled organelles. Two histograms present the results. The first illustrates the size of the organelles where the fraction of labeled organelles was plotted against the area they occupied (Area histogram, Figure 3.33). The second shows the fraction of labeled organelles, which was plotted against the axis ratio (major axis versus the minor axis of the organelles; Ratio histogram, Figure 3.34). The histograms reflect clearly the observed impressions from the three-dimensional reconstructions above. Before dye wash-off (+/- stimulation), most of the labeled organelles occupied large areas (Figure 3.33) and were elongated in shape (Figure 3.34), while at 5 and 30 minutes after stimulation the organelles were small in size (Figure 3.33) and almost round

Figure 3.33: Area histograms of labeled organelles. The histograms show the area the organelles occupied in the different IHCs. The organelles in the zero calcium (orange) and the high potassium stimulation (red) IHCs are larger in size than the organelles of the 5 (blue) and 30 (black) minutes rest IHCs.

Figure 3.34: Axis ratio histograms of labeled organelles. The ratio of the major axis against the minor axis of labeled organelles was calculated and plotted as the fraction of labeled organelles. The axis ratio depicts the nature of the organelles, with spherical objects having a ratio of one while elongated structures (with a larger major axis) exhibit higher ratios. The organelles of the zero calcium IHC (orange) and the stimulated IHC (high potassium, red) are characterized by higher axis ratio values compared to the 5 and 30-minute time points (blue and black, respectively).

The shape of the organelles became also clear when analyzing scatterplots, where the values from the major axis and the minor axis of the organelles were presented (Figure 3.35). Both rested IHCs (5 minutes: blue, 30 minutes: black) showed clustered values with primarily low major and minor axis values, indicating that most of the organelles were small and round shaped. However, the plots of the zero calcium and the stimulated IHCs showed both a broad value distribution, which verified that many types of organelles were present (large and round, large and elliptic, elongated).

Figure 3.35: Scatterplots of major versus minor axis values of labeled organelles. The scatterplots present the nature of the organelles in the different treated IHCs. The “zero calcium” (orange) and the “high potassium” IHCs (red) had broad variations of organelles, with high values for the major and the minor axis. The “5 minutes” (blue) and the “30 minutes” (black) IHCs had organelles with smaller major and minor axis values, indicating small and round organelles.

A second scatterplot analysis represents the area an organelle occupied related to the previously calculated ratio (major axis vs. minor axis) (Figure 3.36). The graphical illustration clearly showed the characteristic morphology of the labeled organelles at the different time points in the recycling pathway. The zero calcium and high potassium stimulated IHCs contained lots of large elongated organelles, as could be seen from the correlation of high area and ratio values. In contrast the 5 minutes and 30 minutes time points were characterized by only small organelles, as large area and ratio values were almost absent. Only few elongated objects were present after 30 minutes. However, they were still smaller in size compared to the organelles from the zero calcium and the stimulation IHCs.

Taken together, the non-stimulated and the stimulated IHCs were both characterized by large elongated and also round organelles, occupying large areas. In contrast, the IHCs at 5 and 30 minutes after stimulation can be described by mostly small spherical organelles.

Figure 3.36: Scatterplots of the area occupied by the organelles versus their axis ratio. The scatterplot shows that the “5 minutes” IHC (blue) had small and round organelles (low area values correlate with low ratio values), similar to the “30 minutes” IHC (black), which had some more elongated tubular structures (low area values correlate with some higher ratio values). The “zero calcium” (orange) and “high potassium” (red) IHCs show both high area values correlating with high ratio values, reflecting the existence of large cisternal organelles.