Decision letter: Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over 30 mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for the development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology and provide new insights into mitochondrial deafness. Editor's evaluation This valuable study of serial block-face scanning electron microscopy on zebrafish lateral line hair cells provided compelling cellular evidence for the importance of normal hair cell function in establishing mitochondrial patterning. This work will be of broad interest to cell biologists studying mitochondrial function. https://doi.org/10.7554/eLife.80468.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Our ability to perceive sounds relies on tiny cells deep inside our ears which can convert vibrations into the electrical signals that our brain is able to decode. These ‘hair cells’ sport a small tuft of short fibers on one of their ends that can move in response to pressure waves. The large amount of energy required for this activity is provided by the cells’ mitochondria, the small internal compartments that act as cellular powerhouses. In fact, reducing mitochondrial function in hair cells can lead to hearing disorders. Mitochondria are often depicted as being bean-like, but they can actually adopt different shapes based on the level of energy they need to produce. Despite this link between morphology and function, little is known about what mitochondria look like in hair cells. Filling this knowledge gap is necessary to understand how these structures support hair cells and healthy hearing. To address this question, McQuate et al. turned to zebrafish, as these animals detect vibrations in water through easily accessible hair cells on their skin that work just like the ones in the mammalian ear. Obtaining and analysing series of 3D images from a high-resolution microscope revealed that hair cells are more densely populated with mitochondria than other cell types. Mitochondrial organisation was also strikingly different. The side of the cell that carries the hair-like structures featured many small mitochondria; however, on the opposite side, which is in contact with neurons, the mitochondria formed a single large network. The co-existence of different types of mitochondria within one cell is a novel concept. Further experiments investigated how these mitochondrial characteristics were connected to hair cell activity. They showed that this organisation was established gradually as the cells aged, with cellular activity shaping the architecture (but not the total volume) of the mitochondria. Overall, the work by McQuate et al. provides important information necessary to develop therapeutics for hearing disorders linked to mitochondrial dysfunction. However, by showing that various kind of mitochondria can be present within one cell, it should also inform studies beyond those that focus on hearing. Introduction Mitochondria are essential subcellular organelles in nearly all eukaryotic cells, where they perform and regulate manifold functions, including ATP production, calcium buffering, apoptosis, metabolite generation, among others. These functions are influenced by a cell’s total mitochondrial volume, regulated by mitochondrial biogenesis (Jornayvaz and Shulman, 2010) and subsequent mitochondrial architecture, sculpted by mitochondrial fusion and fission (Picard et al., 2013). Mitochondrial fusion and elongated mitochondria are associated with heightened mitochondrial membrane potentials, increased ATP production, and improved calcium buffering (Picard et al., 2013; Szabadkai et al., 2006; Gomes et al., 2011). Meanwhile, mitochondrial fission and smaller mitochondria are associated with lower mitochondrial membrane potentials, lower ATP production, and apoptosis (Liu et al., 2020). The combination of these features produces an overall mitochondrial phenotype according to cellular need. Failure to achieve an appropriate mitochondrial phenotype results in a variety of pathologies, particularly in highly metabolically active cells such as those that are electrically excitable (Reddy et al., 2011). Hair cells (HCs) in the peripheral auditory nervous system mediate hearing and balance. Deflection of the stereocilia bundle at the apical pole of the HC results in cation influx in a process known as mechanotransduction, and subsequent depolarization and calcium influx through voltage-gated calcium channels (cav1.3) results in glutamate release from ribbon synapses at the basolateral pole onto afferent neurons. HCs heavily depend on mitochondria to sustain energetic demands, with 75% of their ATP usage produced via oxidative phosphorylation (Puschner and Schacht, 1997). It is perhaps due to their high dependency on mitochondria that HCs are particularly susceptible to mitochondrial alterations; mitochondria are implicated in both hereditary and environmentally induced hearing loss, as well as aging (Kokotas et al., 2007; Someya and Prolla, 2010; Böttger and Schacht, 2013). Mutations in over 30 mitochondrial-associated genes result in hearing loss in humans. These include mutations in the gene opa1, necessary for mitochondrial fusion (Leruez et al., 2013; Liguori et al., 2008). Mitochondria are implicated at both poles of healthy HCs. In rat cochlear inner HCs, apical mitochondria have been shown to buffer calcium influx during mechanotransduction (Beurg et al., 2010; Pickett et al., 2018). Meanwhile, in zebrafish lateral line HCs, basal mitochondrial calcium uptake is essential for regulating ribbon size (Wong et al., 2019). Mitochondria also play a role in HC vulnerability to aminoglycoside exposure. HCs that have been treated with neomycin demonstrate abnormal mitochondrial morphologies prior to other insults (Owens et al., 2007). Neomycin-induced HC death requires mitochondrial calcium uptake (Esterberg et al., 2014 and Esterberg et al., 2016), and HC sensitivity to neomycin increases with cumulative mitochondrial activity (Pickett et al., 2018). Similarly, calcium import into the mitochondria via the MCU has been implicated in noise-induced hearing loss (Wang et al., 2018), and related mitochondrial potentials are disrupted in aging (Perkins et al., 2020). Given the known impact of mitochondrial structure on their function, understanding the detailed morphological characteristics of HC mitochondria is a necessary first step for interpreting how these morphologies intersect with HC physiology and vulnerability (Lesus et al., 2019). We hypothesized that HCs maintained their mitochondria in an optimal configuration dependent on mitochondrial fusion to sustain high metabolic demands. Here, we detailed the characteristics of mitochondria in the HCs of the zebrafish lateral line. The lateral line is composed of clusters of HCs and surrounding supporting cells (SCs) called neuromasts (NMs) found on the surface of the fish’s body and detects changes in water flow. This information is vital for schooling and feeding behaviors. Lateral line HCs are genetically and morphologically similar to the HCs located within the mammalian inner ear, but are more easily accessible to genetic manipulations and experimentations (Pickett and Raible, 2019; Sheets et al., 2021). As fluorescent markers lack the resolution to distinguish between individual organelles, we turned to serial block-face scanning electron microscopy (SBFSEM) to produce three-dimensional reconstructions of HCs and their mitochondria with ultrastructural resolution. Over the course of this study, we reconstructed 5908 individual mitochondria from 162 different cells (16 NMs, 10 fish, Table 1). We demonstrated that zebrafish lateral line HCs had a mitochondrial phenotype distinct from SCs. This phenotype included a high total mitochondrial volume, and a particular architecture, with large, highly networked mitochondria at the basolateral pole near the synaptic ribbons, and smaller mitochondria positioned apically. This phenotype developed with HC maturation and was dependent on mechanotransduction and synaptic activity. Overall, our results demonstrate that HCs developed a highly specialized mitochondrial architecture sculpted by cell activity, which may explain their sensitivity to mitochondrial perturbation. Furthermore, the SBF datasets for each of the NMs used in this study have been made openly available, providing a comprehensive resource for further studying lateral line HCs and their organelles at the ultrastructural level. Table 1 Individual cells and mitochondria reconstructed. GenotypeAge (dpf)FishNeuromastsCellsMitochondriaWT HCs32212299WT HCs5–635652347cdh23 HCs52419382cav1.3 HCs524481939opa1 HCs5115778WT SCs52313163Total1625908 dpf: days post fertilization; HC: hair cell. Results Hair cells contain a dense mitochondrial population not found in supporting cells Although the importance of mitochondria in HCs is well-established, it is unclear how HC mitochondria compare with the mitochondria of other cell types, both in number and morphology. We used SBFSEM to reconstruct HCs and SCs from zebrafish anterior lateral line NMs (Figure 1A). This technique provides sufficient resolution to distinguish between individual mitochondria and compare individual mitochondrial morphologies. HC bodies and HC mitochondria were reconstructed via manual segmentation at 5–6 dpf, an age when the lateral line has completed maturation (Figure 1B, 3 fish, 5 NMs, total of 65 HCs). HCs were distinguishable from other cell types in the NM by the presence of synaptic ribbons, stereocilia, and kinocilia. To compare HC mitochondrial values to those of other cell types, we reconstructed the mitochondria of both peripheral and central SCs (three NMs from two fish, six central and seven peripheral SCs). SCs are a vital component of NMs, both structurally and physiologically. We reconstructed central and peripheral SCs, which have been found to have differing roles within the NM; peripheral SCs symmetrically divide to become HC progenitors during homeostasis and regeneration. Meanwhile, central SCs serve a ‘glial-like’ function in maintaining ion balance (Thomas and Raible, 2019; Romero-Carvajal et al., 2015). Central SCs (Figure 1C) were defined as interdigitating between two HCs, while peripheral SCs (Figure 1D) touched one or no HCs. Figure 1 Download asset Open asset Hair cells (HCs) contain a higher mitochondrial volume than supporting cells. (A) SEM cross-section through 5 days post fertilization (dpf) zebrafish neuromasts (NM) (NM3, Figure 1—source data 2). Scale bar = 20 µm. Inset shows the stereocilia bundle and kinocilium labeled for 1 HC. (B) Six reconstructed HCs from NM3, with mitochondria shown in white. Scale bar = 5 µm. (C) Two central supporting cells (C-SCs) reconstructed from NM3. Scale bar = 5 µm. (D) Two peripheral supporting cells (P-SCs) reconstructed from NM3. Scale bar = 3.5 µm. (E) Sum of mitochondrial volume for HCs, C-SCs, and P-SCs. (In µm3) HC: 14.8 ± 0.8; C-SC; 6.9 ± 1.1, P-SC; 6.0 ± 0.7. (F) Number of individual mitochondria in HCs, C-SCs, and P-SCs. HC: 36.1 ± 1.6; C-SC: 14.5 ± 1.4; P-SC: 10.9 ± 1.5. (G) The median mitochondrial volume in HCs, C-SCs, and P-SCs. HC: 0.2 ± 0.01; C-SC: 0.3 ± 0.04; P-SC: 0.4 ± 0.03. (H) The ratio of the total mitochondrial volume to the total cell volume in HCs, C-SCs, and P-SCs. HC: 0.07 ± 0.003; C-SC: 0.04 ± 0.002; P-SC: 0.04 ± 0.002. (I) The cell volume of HCs, C-SCs, and P-SCs. HC: 195.6 ± 7.2; C-SC: 197.4 ± 30.05; P-SC: 170.4 ± 22.7. Kruskal–Wallis test with Dunn’s multiple comparisons, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For (E–G), HC: n = 65, 5 NMs, 3 fish; C-SC: n = 6, 3 NMs, 2 fish; P-SC: n = 7, 3 NMs, 2 fish. For (H, I), HCs: n = 35, 3 NMs, 3 fish; C-SC: n = 4, 2 NMs, 2 fish; P-SC: n = 4, 2 NMs, 2 fish. Data are presented as the mean ± SEM. Figure 1—source data 1 Raw values used in Figure 1. https://cdn.elifesciences.org/articles/80468/elife-80468-fig1-data1-v2.xlsx Download elife-80468-fig1-data1-v2.xlsx Figure 1—source data 2 Datasets used in Figure 1. https://cdn.elifesciences.org/articles/80468/elife-80468-fig1-data2-v2.docx Download elife-80468-fig1-data2-v2.docx HCs contained on average a total mitochondrial volume of 14.8 ± 0.8 µm3 distributed across 36.1 ± 1.6 individual mitochondria (Figure 1E and F). The HC median mitochondrion volume was 0.2 ± 0.01 µm3 (Figure 1G). The average ratio of HC mitochondrial volume to cell volume was approximately 7% (Figure 1H). Both types of SCs had less total mitochondrial volume than HCs. We found central SCs contained a total mitochondrial volume of 6.9 ± 1.1 µm3 distributed over 14.5 ± 1.4 individual mitochondria (Figure 1E and F), with a median mitochondrial volume of 0.3 ± 0.04 µm3 (Figure 1G). Similarly, peripheral SCs contained a total mitochondrial volume of 6.0 ± 0.7 µm3 distributed over 10.9 ± 1.6 individual mitochondria with a median mitochondrial volume of 0.4 ± 0.03 µm3 (Figure 1E–G). In both central and peripheral SCs, the ratio of mitochondrial volume to cell volume averaged around 4% (Figure 1H). By contrast, overall cell volumes of HCs and both SC types were not different (Figure 1I). These data demonstrate that HCs have elevated mitochondrial volume and number relative to SCs. Mitochondrial architecture develops with hair cell maturation We next asked how HC mitochondria change during cellular maturation. In the zebrafish lateral line, HCs undergo homeostatic turnover within NMs, where older, dying HCs are replaced when HC progenitors symmetrically divide to produce two new daughter HCs. As a result, NMs contain a spectrum of HCs of different ages. We used the length of the tallest stereocilium (stereocilia length) and length of the kinocilium to approximate the age of each individual HC as these both grow longer as HCs mature (Kindt et al., 2012). The actin-based stereocilia bundle contains HC tip links and mechanotransductive channels. The kinocilium is a microtubule-based structure that in the zebrafish lateral line has a role in HC development and establishing mechanotransduction. While some mammalian HCs shed their kinocilium, in zebrafish HCs the kinocilium continues to grow throughout the cell’s lifespan. The lengths of both the stereocilia and kinocilium confirmed that the HCs we analyzed span the range of development (Figure 2—figure supplement 1A and B). The height of the stereocilia bundle and length of kinocilium also demonstrated a significant, positive correlation (Figure 2—figure supplement 1C). These metrics can then be used to relate HC age and HC mitochondrial properties. As HCs mature, their mitochondria increase in volume and complexity (Figure 2A and A’). We found a positive correlation between HC stereocilia length and total mitochondrial volume (Figure 2B, p<0.0001) and the number of individual mitochondria (Figure 2C, p=0.003). Similar trends were found when kinocilium length was used to approximate HC age (Figure 2—figure supplement 1D and E). Because complete stereocilia bundles were more readily preserved than complete kinocilia over SBF serial sectioning, we focused on stereocilia length for the remainder of this study. These observations demonstrate that mitochondrial number and volume continue to grow as HCs mature. Figure 2 with 2 supplements see all Download asset Open asset Hair cells (HCs) gradually develop a high mitochondrial volume with specific architecture. (A) Two young HCs from a 5 days post fertilization (dpf) neuromasts (NM) (NM1, Figure 2—source data 2). Mitochondria are shown in white. Single largest mitochondrion (max mito) is shown in gold. Synaptic ribbons shown in purple. Scale bar = 5 µm. (A’) Two mature HCs from a 5 dpf NM (NM3, different HCs than in Figure 1B). Scale bar = 6 µm. (B) Relationship between HC stereocilia length and the total mitochondrial volume. (C) Relationship between HC stereocilia length and the number of individual mitochondria. (D) Relationship between HC stereocilia length and the volume of the median mitochondrion. (E) Relationship between HC stereocilia length and the volume of the largest mitochondrion (max mito). (F) Relationship between HC stereocilia length and the number of standard deviations between the max mito and average mitochondrial volume (max mito z-score). Lines represent standard linear regression, with significance as indicated. (G) The z-score of the max mito in HCs, central supporting cells (C-SCs), and peripheral supporting cells (P-SCs) (mean ± SEM) HC: 4.1 ± 0.1; C-SC: 2.4 ± 0.1, P-SC: 2.1 ± 0.2. Kruskal–Wallis test with Dunn’s multiple comparisons, ***p<0.001. (H) The percentage of the max mito located within each quadrant of an HC represented as a heat map. The length of each HC was normalized and broken into quadrants, with the highest HC point the base of the stereocilia bundle and the lowest point the lowest ribbon. The number of max mito segmentation coordinates within each quadrant of an HC were counted and represented as a percentage of all max mito coordinates. Cells are presented in order of their stereocilia lengths. (I) Summary of heat map data shown in (H). Most apical quadrant (0–25%): 9 ± 2.4%; 25–50%: 17.1 ± 2.7%; 50–75%: 21 ± 2.2%; Most basal quadrant (75–100%): 52.8 ± 4.4%. Kruskal–Wallis test with Dunn’s multiple comparisons, **p<0.01, ****p<0.0001. (J) Percentage of the max mito located within the most basal quadrant for individual HCs. HC: n = 65, 5 NMs, 3 fish; C-SC: n = 6, 3 NMs, 2 fish; P-SC: n = 7, 3 NMs, 2 fish. Figure 2—source data 1 Raw values used in Figure 2. https://cdn.elifesciences.org/articles/80468/elife-80468-fig2-data1-v2.xlsx Download elife-80468-fig2-data1-v2.xlsx Figure 2—source data 2 Datasets used in Figure 2. https://cdn.elifesciences.org/articles/80468/elife-80468-fig2-data2-v2.docx Download elife-80468-fig2-data2-v2.docx Next, we examined the uniformity of the HC mitochondrial population. We found that over the course of maturation, HCs often developed a single, large, interconnected mitochondrion (max mito, Figure 2A’) that steadily expanded in volume (Figure 2E). In contrast, the volume of the median mitochondrion stayed relatively constant over HC development (Figure 2D). Therefore, the number of standard deviations between the max mito volume and the mean mitochondrial volume (max mito z-score) steadily increased during HC maturation (Figure 2F). By comparison, the mitochondrial populations of both central and peripheral SCs were more homogenous (Figure 2G). The distribution of individual mitochondrion volumes for representative mature HCs is shown in Figure 2—figure supplement 2. As HCs mature, the max mito localized to the basolateral pole of the HC, such that on average 53% of its total volume was localized in the bottom-most quadrant of the HC (Figure 2H–J). These data show that as HCs develop, the mitochondrial population becomes nonuniform, with smaller mitochondria positioned apically, and a max mito localized to the base of the HC. Ribbon growth parallels mitochondrial growth and localization HC mitochondria are known to regulate ribbon volume, and their ability to buffer calcium (Wong et al., 2019) and to generate ATP (Stowers et al., 2002; Perkins et al., 2010) at the basal end of HCs suggest they have roles in synaptic transmission. We therefore asked whether there was a relationship between HC mitochondrial development and ribbon development. We first measured ribbon volume and number across HC development. Averaging across all ages, HCs contained 5.5 ± 0.2 ribbons, with an average ribbon volume of 0.1 ± 0.006 µm3, for a total ribbon volume of 0.6 ± 0.03 µm3 (Figure 3—figure supplement 1A–C). The total ribbon volume steadily expanded over HC maturation (Figure 3A and B). The increase in total ribbon volume was primarily attributed to an increase in individual ribbon volume (Figure 3C) as there was no significant change in ribbon number over maturation (Figure 3D). We next compared ribbon development to mitochondrial development. We found a strong, positive correlation between the total mitochondrial volume and the total ribbon volume of each HC (Figure 3E). Given the localization of the max mito to the basolateral pole, we then asked whether the max mito was specifically associated with synaptic ribbons by calculating the average minimum geometric distance between the max mito and each ribbon per HC. We found that during HC maturation, the max mito localized to the synaptic ribbons, as reflected in the nonlinear decrease in the average minimum distance (Figure 3F). Meanwhile, there was no change in the position of the median mitochondrion relative to the ribbons during HC maturation (Figure 3G). Averaging across all HCs, the max mito was consistently closer to the synaptic ribbons (average, 1.9 ± 0.3 µm) than the median mitochondrion (6.3 ± 0.3 µm) (Figure 3H). These data show that during HC maturation, the size of individual ribbons increases in tandem with mitochondrial volume, and that the max mito becomes increasingly associated with ribbon synapses. Figure 3 with 1 supplement see all Download asset Open asset Ribbon growth parallels mitochondrial growth and localization. (A) Two representative young hair cells (HCs) from a 6 days post fertilization (dpf) neuromasts (NM) (NM4, Figure 3—source data 2) with synaptic ribbons shown in purple. Scale bar = 4.5 µm. (A’) Two representative mature HCs from a 6 dpf NM (NM4, Figure 3—source data 2). Scale bar = 4 µm. (B) Relationship between HC stereocilia length and the total ribbon volume. (C) Relationship between HC stereocilia length and the average ribbon volume. (D) Relationship between HC stereocilia length and the number of ribbons. (E) Relationship between HC total mitochondrial volume and HC ribbon volume. Black line = standard linear regression, with significance as indicated. (F) Relationship between HC stereocilia length and the average minimum distance between each ribbon and the max mito. (G) Relationship between HC stereocilia length and the average minimum distance between each ribbon and the median mito. (H) Average minimum distance between each ribbon and the HC max or median mito. (In µm) Max mito: 1.9 ± 0.3; median mito: 6.3 ± 0.3. Mann–Whitney test, ****p<0.0001. HC: n = 65, 5 NMs, 3 fish, 5–6 dpf. Figure 3—source data 1 Raw values used in Figure 3. https://cdn.elifesciences.org/articles/80468/elife-80468-fig3-data1-v2.xlsx Download elife-80468-fig3-data1-v2.xlsx Figure 3—source data 2 Datasets used in Figure 3. https://cdn.elifesciences.org/articles/80468/elife-80468-fig3-data2-v2.docx Download elife-80468-fig3-data2-v2.docx Maturity of the neuromast affects mitochondrial architecture To further explore patterns of mitochondrial maturation with HC development, we next examined HCs in immature 3 dpf NMs (2 NMs, 2 fish, total 12 HCs, Figure 4A), which, while shown to be functional, primarily demonstrate young biophysical profiles (Olt et al., 2014). Consistent with this idea, stereocilia length of 3 dpf HCs were on average shorter than but fell within the range of those from 5 to 6 dpf HCs (Figure 4—figure supplement 1). Similar to 5–6 dpf HCs, 3 dpf HCs gained mitochondrial volume with increasing stereocilia length (Figure 4A, A’ and B), with an average total mitochondrial volume of 14.4 ± 1.2 µm3 (Figure 4B). Cell volume and ratio of mitochondrial volume to cell volume were not different between 3 dpf HCs and 5–6 dpf HCs (Figure 4C). 3 dpf HCs had on average fewer mitochondria (24.9 ± 2 individual mitochondria) than 5–6 dpf HCs (Figure 4D). In tandem, the median mitochondrion volume in 3 dpf HCs (0.3 ± 0.02 µm3) remained larger than that of 5–6 dpf HCs, regardless of HC age (Figure 4E). Although the volume of the max mito on average was the same between 3 dpf and 5–6 dpf HCs, and the z-score was unaffected, it did not appear to significantly gain volume (Figure 4F and G). Additionally, the max mito did not localize to the basolateral pole, but remained randomly distributed throughout the HC (Figure 4H–J). 3 dpf HCs also had smaller ribbons (0.06 ± 0.007 µm3) and lower total ribbon volumes (0.35 ± 0.04 µm3) than 5–6 dpf HCs, though the number of ribbons (6 ± 0.4) was unaffected ( Figure 4—figure supplement 2A–C). The localization of the max mito to ribbons at 3 dpf was less than at 5–6 dpf (Figure 4—figure supplement 2E and F), though the relationship between mitochondrial volume and ribbon volume remained unaltered (Figure 4—figure supplement 2). These data suggest that while the HC mitochondrial volume expands independent of HC biophysical properties, development of proper mitochondrial architecture follows maturation of the NM. Figure 4 with 2 supplements see all Download asset Open asset 3 days post fertilization (dpf) hair cells (HCs) demonstrate immature mitochondrial architecture. (A) Representative young HCs from a 3 dpf neuromasts (NM) (NM6, Figure 4—source data 2). Max mito shown in gold. Synaptic ribbons shown in purple. Scale bar = 8 µm. (A’) Representative older HCs from a 3 dpf NM (NM6, Figure 4—source data 2). Scale bar = 6 µm. (B) Comparison of total mitochondrial volume development (left) and average (right) between 3 dpf and 5–6 dpf HCs. On average (in µm3): 3 dpf: 14.4 ± 1.2; 5–6 dpf: 14.8 ± 0.8. Kolmogorov–Smirnov test, p=0.49. (C) Total HC volume for 3 dpf and 5–6 dpf HCs. (In µm3) 3 dpf: 210.1 ± 11.3; 5–6 dpf: 195.6 ± 7.2. Kolmogorov–Smirnov test, p=0.2. (C’) Ratio of total mitochondrial volume to HC volume. 3 dpf: 0.06 ± 0.003; 5–6 dpf: 0.07 ± 0.003. Kolmogorov–Smirnov test, p=0.41. (D) Comparison of the number of HC mitochondria over development (left) and on average (right) in 3 dpf and 5–6 dpf HCs. On average: 3 dpf: 25.0 ± 2; 5–6 dpf: 36.1 ± 1.6. Kolmogorov–Smirnov test, p=0.022. (E) Comparison of the median mitochondrial volume over development (left) and on average (right). On average (in µm3): 3 dpf: 0.3 ± 0.02; 5–6 dpf: 0.2 ± 0.01. Kolmogorov–Smirnov test, p=0.005. (F) Comparison of the max mito volume over development (left) and on average (right). On average (in µm3): 3 dpf: 3.8 ± 0.5; 5–6 dpf: 3.5 ± 0.4. Kolmogorov–Smirnov test, p=0.32. (G) Comparison of the max mito z-score in 3 dpf and 5–6 dpf HCs over development (left) and on average (right). On average: 3 dpf: 3.7 ± 0.3; 5–6 dpf: 4.1 ± 0.1. Standard unpaired t-test, p=0.21. (B–G) Solid line represents the standard linear regression for 3 dpf HCs. Dashed line represents standard regression for the 5–6 dpf HCs dataset as in Figure 2. Significance of the 3 dpf regression and difference with 5–6 dpf regression slope (S) are indicated. (H) The percentage of the max mito located within each quadrant of 3 dpf HCs represented as a heat map. Two HCs in the 3 dpf dataset lacked ribbons to provide a consistent HC lowest point and were not included in this analysis. (I) Summary of the heat map data shown in (H). Most apical quadrant (0–25%): 24.9 ± 8.9%; 25–50%: 37.2 ± 5.7%; 50–75%: 16.0 ± 4.2%; Most basal quadrant (75–100%): 22.0 ± 7.9%. Kruskal–Wallis test with Dunn’s multiple comparisons, nonsignificant. (J) Percentage of max mito located within the most basal quadrant for individual HCs. 3 dpf: 22.0 ± 7.9%, 5–6 dpf: 52.8 ± 4.4%. Kolmogorov–Smirnov test, p=0.017. Same cells as in (H, I). (B, D–G) 3 dpf data: n = 12 HCs, 2 NMs, 2 fish. 5–6 dpf data: n = 65 HCs, 5 NMs, 3 fish. (C, C’) 3 dpf data: n = 12 HCs, 2 NMs, 2 fish. 5–6 dpf data: n = 35 HCs, 3 NMs, 3 fish. (H–J) 3 dpf data: n = 10 HCs, 2 NMs, 2 fish. 5–6 dpf data: n = 65 HCs, 5 NMs, 3 fish. Where applicable, data are presented as the mean ± SEM. Figure 4—source data 1 Raw values used in Figure 4. https://cdn.elifesciences.org/articles/80468/elife-80468-fig4-data1-v2.xlsx Download elife-80468-fig4-data1-v2.xlsx Figure 4—source data 2 Datasets used in Figure 4. https://cdn.elifesciences.org/articles/80468/elife-80468-fig4-data2-v2.docx Download elife-80468-fig4-data2-v2.docx Disrupting hair cell mitochondrial architecture impacts mitochondrial calcium buffering To test the impact of mitochondrial architecture on mitochondrial function, we created a CRISPR mutant for the gene opa1, a conserved dynamin-like GTPase necessary for the fusion of the inner mitochondrial membrane for which loss of function results in mitochondrial fragmentation from yeast to humans (Oli