Ling Huang
email: ling.huang@hds.utc.fr

Thierry Denoeux

Pierre Vera

Su Ruan

Evidence fusion

Keywords: Information fusion, Dempster-Shafer theory, Evidence theory, Uncertainty quantification, Contextual discounting, Deep learning, Brain tumor segmentation

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Evidence fusion with contextual discounting for multi-modality medical image segmentation ⋆

Introduction

Single-modality medical images often do not contain enough information to reach a reliable diagnosis. This is why physicians generally use multiple sources of information, such as multi-MR images for brain tumor segmentation, or PET-CT images for lymphoma segmentation. The effective fusion of multi-modality information is of great importance in the medical domain for better disease diagnosis and radiotherapy. Using convolutional neural networks (CNNs), researchers have mainly adopted probabilistic approaches to information fusion, which can be classified into three strategies: image-level fusion such as input data concatenation [START_REF] Peiris | A volumetric transformer for accurate 3d tumor segmentation[END_REF], feature-level fusion such as attention mechanism concatenation [START_REF] Zhou | Fusion based on attention mechanism and context constraint for multi-modal brain tumor segmentation[END_REF], and decision-level fusion such as weighted averaging [START_REF] Kamnitsas | Ensembles of multiple models and architectures for robust brain tumour segmentation[END_REF]. However, probabilistic fusion is unable to effectively manage conflict that occurs when different labels are assigned to the same voxel based on different modalities. Also, it is important, when combining multi-modality information, to take into account the reliability of the different sources [START_REF] Mercier | Refined modeling of sensor reliability in the belief function framework using contextual discounting[END_REF].

Dempster-Shafer theory (DST) [START_REF] Dempster | Upper and lower probability inferences based on a sample from a finite univariate population[END_REF][20], also known as belief function theory or evidence theory, is a formal framework for information modeling, evidence combination, and decision-making with uncertain or imprecise information [START_REF] Denoeux | Representations of uncertainty in artificial intelligence: Beyond probability and possibility[END_REF]. Researchers from the medical image community have actively investigated the use of DST for handling uncertain, imprecision sources of information in different medical tasks, such as disease classification [START_REF] Huang | Covid-19 classification with deep neural network and belief functions[END_REF], tumor segmentation [START_REF] Huang | Evidential segmentation of 3D PET/CT images[END_REF] [START_REF] Huang | Lymphoma segmentation from 3D PET-CT images using a deep evidential network[END_REF], multi-modality image fusion [START_REF] Huang | Deep PET/CT fusion with Dempster-Shafer theory for lymphoma segmentation[END_REF], etc. In the DST framework, the reliability of a source of information can be taken into account using the discounting operation [START_REF] Shafer | A mathematical theory of evidence[END_REF], which transforms each piece of evidence provided by a source into a weaker, less informative one.

In this paper, we propose a multi-modality evidence fusion framework with contextual discounting based on DST and deep learning. To our knowledge, this work is the first attempt to apply evidence theory with contextual discounting to the fusion of deep neural networks. The idea of considering multi-modality images as independent inputs and quantifying their reliability is simple and reasonable. However, modeling the reliability of sources is important and challenging. Our model computes mass functions assigning degrees of belief to each class and an ignorance degree to the whole set of classes. It thus has one more degree of freedom than a probabilistic model, which allows us to model source uncertainty and reliability directly. The contributions of this paper are the following:

(1) Four DST-based evidential segmentation modules are used to compute the belief of each voxel belonging to the tumor for four modality MRI images; (2) An evidence-discounting mechanism is applied to each of the single-modality MRI images to take into account its reliability; (3) A multi-modality evidence fusion strategy is then applied to combine the discounted evidence with DST and achieve more reliable results. End-to-end learning is performed by minimizing a new loss function based on the discounting mechanism, allowing us to increase the segmentation performance and reliability.

The overall framework will first be described in Section 2 and experimental results will be reported in Section 3.

Methods

Fig. 1 summarizes the proposed evidence fusion framework with contextual discounting. It is composed of (1) four encoder-decoder feature extraction (FE) modules (Residual-UNet [START_REF] Kerfoot | Left-ventricle quantification using residual u-net[END_REF]), (2) four evidential segmentation (ES) modules, and (3) a multi-modality evidence fusion (MMEF) module. Details about the ES and MMEF modules will be given in Sections 2.1 and 2.2, respectively. The discounted loss function used for optimizing the framework parameters will be described in Section 2.3.

Evidential Segmentation

Dempster-Shafer theory. Before presenting the evidential segmentation module, we first introduce some basic concepts of DST. Let Ω = {ω 1 , ω 2 , . . . , ω K } be a finite set of hypotheses about some question. Evidence about Ω can be represented by a mapping m from 2 Ω to [0, 1] such that A⊆Ω m(A) = 1, called a mass function. For any hypothesis A ⊆ Ω, the quantity m(A) represents the mass of belief allocated to A and to no more specific proposition. The associated belief and plausibility functions are defined, respectively, as

Bel(A) = ∅̸ =B⊆A m(B) and P l(A) = B∩A̸ =∅ m(B), ∀A ⊆ Ω. (1)
The contour function pl associated to m is the function that maps each element ω of Ω to its plausibility: pl(ω) = P l({ω}).

Two mass functions m 1 and m 2 derived from two independent items of evidence can be combined by Dempster's rule [START_REF] Shafer | A mathematical theory of evidence[END_REF], denoted as ⊕ and formally defined by (m 1 ⊕ m 2 )(∅) = 0 and

(m 1 ⊕ m 2 )(A) = 1 1 -κ B∩C=A m 1 (B)m 2 (C), (2)
for all A ⊆ Ω, A ̸ = ∅, where κ represents the degree of conflict between m 1 and m 2 :

κ = B∩C=∅ m 1 (B)m 2 (C). (3)
The combined mass m 1 ⊕ m 2 is called the orthogonal sum of m 1 and m 2 . Let pl 1 , pl 2 and pl 12 denote the contour functions associated with, respectively, m 1 , m 2 and m 1 ⊕ m 2 . the following equation holds:

pl 12 = pl 1 pl 2 1 -κ . (4)
Equation ( 4) allows us to compute the contour function in time proportional to the size of Ω, without having to compute the combined mass m 1 ⊕ m 2 .

Evidential segmentation module Based on the evidential neural network model introduced in [START_REF] Denoeux | A neural network classifier based on Dempster-Shafer theory[END_REF] and using an approach similar to that recently described in [START_REF] Huang | Evidential segmentation of 3D PET/CT images[END_REF], we propose a DST-based evidential segmentation module to quantify the uncertainty about the class of each voxel by a mass function. The basic idea of the ES module is to assign a mass to each of the K classes and to the whole set of classes Ω, based on the distance between the feature vector of each voxel and I prototype centers. The input feature vector can be obtained using any FE module, e.g., Residual-UNet [START_REF] Kerfoot | Left-ventricle quantification using residual u-net[END_REF], nnUNet [START_REF] Isensee | nnu-net: Selfadapting framework for u-net-based medical image segmentation[END_REF].

The ES module is composed of an input layer, two hidden layers and an output layer. The first input layer is composed of I units, whose weights vectors are prototypes p 1 , . . . , p I in input space. The activation of unit i in the prototype layer for input vector x is

s i = α i exp(-γ i ∥x -p i ∥ 2 ), (5)
where γ i > 0 and α i ∈ [0, 1] are two parameters. The second hidden layer computes mass functions m i representing the evidence of each prototype p i using the following equations:

m i ({ω k }) = u ik s i , k = 1, ..., K (6a)
m i (Ω) = 1 -s i , (6b)
where u ik is the membership degree of prototype i to class ω k , and K k=1 u ik = 1. Finally, the third layer combines the I mass functions m 1 , . . . , m I using Dempster's combination rule [START_REF] Bakas | Advancing the cancer genome atlas glioma mri collections with expert segmentation labels and radiomic features[END_REF] to finally obtain a belief function quantifying uncertainty about the class of each voxel.

Multi-modality evidence fusion

In this paper, the problem of quantifying the source reliability is addressed by the discounting operation of DST. Let m be a mass function on Ω and β be a coefficient in [0, 1]. The discounting operation [START_REF] Shafer | A mathematical theory of evidence[END_REF] with discount rate 1 -β transforms m into a weaker, less informative mass function β m defined as

β m = β m + (1 -β) m ? , (7)
where m ? is the vacuous mass function defined by m ? (Ω) = 1, and coefficient β is the degree of belief that the source mass function m is reliable [START_REF] Smets | The Transferable Belief Model[END_REF]. When β = 1 we accept mass function m provided by the source and take it as a description of our knowledge; when β = 0, we reject it and we are left with the vacuous mass function m ? . In this paper, we focus on the situation when β ∈ [0, 1] and combine uncertain evidence with partial reliability using Dempster's rule. As suggested in [START_REF] Mercier | Refined modeling of sensor reliability in the belief function framework using contextual discounting[END_REF], the above discounting operation can be extended to contextual discounting. This operation can represent richer meta-knowledge regarding the reliability of the source of information in different contexts [START_REF] Denoeux | A new evidential k-nearest neighbor rule based on contextual discounting with partially supervised learning[END_REF]. It is parameterized by a vector β = (β 1 , . . . , β K ), where β k is the degree of belief that the source is reliable given that the true class is ω k . The complete expression of the discounted mass function is given in [START_REF] Mercier | Refined modeling of sensor reliability in the belief function framework using contextual discounting[END_REF]. Here, we just give the expression of the corresponding contour function, which will be used later:

β pl({ω k }) = 1 -β k + β k pl({ω k }), k = 1, . . . , K, (8)
When we have several sources provided by independent evidence, the discounted evidence can be combined by Dempster's rule. Assuming that we have two source of information, let β 1 pl S1 and β 2 pl S2 be the discounted contour functions provided, respectively, by sources S 1 and S 2 , with discount rate vectors 1 -β 1 and 1 -β 2 . From (4), the combined contour function is proportional to the product β 1 pl S1 β 2 pl S2 .

Discounted Dice loss

We define a new loss function based on discount rates, hereafter referred to as the discounted Dice loss, given as follows:

loss D = 1 - 2 N n=1 β S n G n N n=1 β S n + N n=1 G n , (9a)
where β S n is the normalized segmentation output for voxel n by fusing H discounted source information,

β S n = H h=1 β h pl S h ({ω k }) K k=1 H h=1 β h pl S h ({ω k }) , (9b)
G n is the ground truth for voxel n, and N is the number of voxels in the volume.

Experimental results

In this section, we report results of our methods on the brain tumor segmentation dataset. The experimental settings will be described in Section 3.1. A comparison with the state of the art and an ablation study will be reported in Section 3.2.

Experiment settings

BraTS Dataset We used 1251 MRI scans with size 240 × 240 × 155 voxels coming from the BraTS 2021 Challenge [START_REF] Baid | The rsna-asnr-miccai brats 2021 benchmark on brain tumor segmentation and radiogenomic classification[END_REF] [16] [START_REF] Bakas | Advancing the cancer genome atlas glioma mri collections with expert segmentation labels and radiomic features[END_REF] to evaluate our framework. For each scan, there are four modalities: native (T1), post-contrast T1weighted (T1Gd), T2-weighted (T2), and T2 Fluid Attenuated Inversion Recovery (FLAIR). Annotations of 1251 scans comprise the GD-enhancing tumor (ET-label 4), the peritumoral edema (ED-label 2), and the necrotic and non-enhancing tumor core (NCR/NET-label1). The task is to segment three semantically meaningful tumor classes: the Enhancing Tumor (ET), the Tumor Core (TC) region (ET+NCR/NET), and the Whole Tumor (WT) region (ET+NCR/NET+ED). Following [START_REF] Peiris | A volumetric transformer for accurate 3d tumor segmentation[END_REF], we divided the 1251 scans into 834, 208, 209 scans for training, validation and testing, respectively. Using the same preprocessing operation as in [START_REF] Peiris | A volumetric transformer for accurate 3d tumor segmentation[END_REF], we performed min-max scaling operation followed by clipping intensity values to standardize all volumes, and cropped/padded the volumes to a fixed size of 128 × 128 × 128 by removing unnecessary background.

Implementation details

The initial values of parameters α i and γ i were set, respectively, to 0.5 and 0.01, and membership degrees u ik were initialized randomly by drawing uniform random numbers and normalizing. The prototypes were initialized by the k-means clustering algorithm. Details about the initialization of ES module parameters can be found in [START_REF] Huang | Lymphoma segmentation from 3D PET-CT images using a deep evidential network[END_REF]. For the MMEF module, the initial values of parameter β k was set to 0.5. Each model was trained on the learning set with 100 epochs using the Adam optimization algorithm. The initial learning rate was set to 10 -3 . The model with the best performance on the validation set was saved as the final model for testing5 .

Segmentation Results

Segmentation Accuracy We used the Dice Score and the Hausdorff Distance (HD) as our evaluation metrics. For each patient, we separately computed these two indices for three classes and then averaged indices over the patients, following a similar evaluation strategy as in [START_REF] Peiris | A volumetric transformer for accurate 3d tumor segmentation[END_REF]. We used two baseline models, Residual-UNet [START_REF] Kerfoot | Left-ventricle quantification using residual u-net[END_REF] and nnUNet [START_REF] Isensee | nnu-net: Selfadapting framework for u-net-based medical image segmentation[END_REF], as FE modules and applied our proposal based on the two corresponding models to construct our methods named, respectively, MMEF-UNet and MMEF-nnUNet. We compared our methods with two recent transformer-based models (nnFormer [START_REF] Zhou | nnformer: Interleaved transformer for volumetric segmentation[END_REF], VT-UNet [START_REF] Peiris | A volumetric transformer for accurate 3d tumor segmentation[END_REF]), two classical CNN-based methods (UNet [START_REF] Abdulkadir | 3d u-net: learning dense volumetric segmentation from sparse annotation[END_REF], V-Net [START_REF] Milletari | V-net: Fully convolutional neural networks for volumetric medical image segmentation[END_REF]), and the two baseline methods. The quantitative results are reported in Table 1. Our methods outperform the two classical CNN-based models and two recent transformer-based methods according to the Dice score, the best result being obtained by MMEF-nnUNet according to this criterion. In contrast, MMEF-UNet achieves the lowest HD.

Segmentation Reliability To test the reliability of our methods, we computed the Expected Calibration Error (ECE) [START_REF] Guo | On calibration of modern neural networks[END_REF] for the two baseline methods and our methods. We obtained ECE values of 2.35 % and 2.04%, respectively, for Residual-UNet and MMEF-UNet, against ECE values of 4.46% and 4.05%, respectively, for nnUNet and MMEF-nnUNet, respectively. The probabilities computed by our models thus appear to be better calibrated.

Ablation Study We also investigated the contribution of each module component to the performance of the system. Table 2 highlights the importance of introducing the ES and MMEF modules. Residual-UNet is the baseline model that uses the softmax transformation to map feature vectors to probabilities. Compared to Residual-UNet, Residual-UNet-ES uses the ES module instead of softmax. Residual-UNet-ES-MMEF, our final proposal, fuses the four single modality outputs from Residual-UNet-ES with MMEF module. Compared to the baseline method Residual-UNet, our method, which plugs the ES module after Residual-UNet, improves the segmentation performance based on single T1Gd, T1 and Flair inputs. Furthermore, the use of the MMEF module improves the performance to a large amount compared to any single modality.

Interpretation of reliability coefficients Table 3 shows the learnt reliability values β for the four modalities with three different classes. The evidence from the T1Gd modality is reliable for ET, ED and NRC classes with the highest β value. In contrast, the evidence from the Flair modality is only reliable for the ED class, and the reliability value β of the T2 modality is only around 0.4 for three classes. The evidence from the T1 modality is less reliable for the three classes compared to the evidence of the other three modalities. These reliability results are consistent with domain knowledge about these modalities reported in [START_REF] Baid | The rsna-asnr-miccai brats 2021 benchmark on brain tumor segmentation and radiogenomic classification[END_REF]. Fig. 2 shows the segmentation results of Residual-UNet with the inputs of four concatenated modalities and MMEF-UNet with the inputs of four separate modalities. Our model locates and segments brain tumors precisely, especially the ambiguous voxels located at the tumor boundary.

Conclusion

Based on DST, a multi-modality evidence fusion framework considering segmentation uncertainty and source reliability has been proposed for multi-MRI brain tumor segmentation. The ES module performs tumor segmentation and uncertainty quantification, and the MMEF module allows for multi-modality evidence fusion with contextual discounting and Dempster's rule. This work is the first to implement contextual discounting for the fusion of multi-modal information with DST and DNN. The contextual discounting operation allows us to take into account the uncertainty of the different sources of information directly, and it reveals the reliability of different modalities in different contexts. Our method can be used together with any state-of-the-art FE module to get better performance. Some limitations of this work remain in the computation cost and the segmentation accuracy. We treat single modality images as independent inputs using independent FE and ES modules, which introduces additional computation costs compared to image concatenation methods (e.g., the FLOPs and parameter numbers are equal to 280.07G and 76.85M for UNet-MMEF, against 73.32G,

Fflair images

Residual-UNet MMEF-Unet Ground Truth Fig. 2. Visualized segmentation results. The first and the second row are the whole brain with tumor and the detailed tumor region (the main differences are marked in blue circles). The three columns correspond, from left to right, to the Flair image, the ground truth and the segmentation results obtained by Residual-UNet and MMEF-UNet. The green, yellow and red represent the ET, ED and NRC/NET, respectively. and 19.21M for Residual-UNet). In future research, we will refine algorithm to improve the accuracy and reliability of our model, and reduce its complexity. We will also explore the possibility of cross-modality evidence fusion for survival prediction tasks.

Fig. 1 .

 1 Fig. 1. Multi-modality evidence fusion framework. It is composed of four encoderdecoder feature extraction (FE) modules corresponding to T1Gd, T1, T2 and Flair modality inputs; four evidential segmentation (ES) modules corresponding to each of the inputs; and a multi-modality evidence fusion (MMEF) module.

Table 1 .

 1 Segmentation Results on the BraTS 2021 dataset. The best result is shown in bold, and the second best is underlined. Ours) 86.96 87.46 90.68 88.36 10.20 6.07 5.29 7.18 MMEF-nnUNet (Ours) 87.26 90.05 92.83 90.05 10.09 9.68 5.10 8.29

	Methods		Dice score	Hausdorff distance
		ET	TC	WT	Avg ET	TC WT Avg
	UNet [3]	83.39 86.28 89.59 86.42 11.49 6.18 6.15 7.94
	VNet [18]	81.04 84.71 90.32 85.36 17.20 7.48 7.53 10.73
	nnFormer [22]	82.83 86.48 90.37 86.56 11.66 7.89 8.00 9.18
	VT-UNet [19]	85.59 87.41 91.20 88.07 10.03 6.29 6.23 7.52
	Residual-UNet [15]	85.07 87.61 89.78 87.48 11.76 6.14 6.31 8.07
	nnUNet [13]	87.12 90.31 91.47 89.68 12.46 11.04 5.97 9.82
	MMEF-UNet (

Table 2 .

 2 Segmentation Results on BraTS 2021 Data (↑ means higher is better).

	Methods	Input Modality			Dice
			ET	ED	NCR/NET Avg
	Residual-UNet	T1Gd	84.13 ↑ 67.44 72.64	74.73
	Residual-UNet-ES	T1Gd	83.92 68.34 ↑ 73.29↑	75.18↑
	Residual-UNet	T1	53.67 63.51 42.81	53.33
	Residual-UNet-ES	T1	57.26↑ 67.83↑ 54.77↑	59.95↑
	Residual-UNet	T2	55.49↑ 69.44 51.16	58.70↑
	Residual-UNet-ES	T2	54.96 69.84↑ 51.27↑	58.69
	Residual-UNet	Flair	50.48 75.16 38.06	54.56
	Residual-UNet-ES	Flair	53.67↑ 77.22↑ 49.71↑	60.20↑
	Residual-UNet-ES-MMEF T1Gd,T1,T2,Flair 86.96↑ 85.48↑ 78.98↑	83.81↑

Table 3 .

 3 Reliability value β (after training) for classes ET, ED and NRC/NET and the four modalities. Higher values correspond to greater contribution to the segmentation.

	β	ET	ED	NRC/NET
	T1Gd 0.9996 0.9726	0.9998
	T1	0.4900 0.0401	0.2655
	T2	0.4814 0.3881	0.4563
	Flair 0.0748 0.86207 0.07512

Our code will be available at https://github.com/iWeisskohl.

⋆ This work was supported by the China Scholarship Council (No. 201808331005). It was carried out in the framework of the Labex MS2T, which was funded by the French Government, through the program "Investments for the future" managed by the National Agency for Research (Reference ANR-11-IDEX-0004-02)


Linda Bieniussa

Montserrat Ipsa Jain

Montserrat Bosch Grau

Lukas Juergens

Rudolf Hagen

Carsten Janke

Kristen Rak
email: rak_k@ukw.de

; Jain

; Juergens

; Hagen

; Janke

Carsten

; Rak

Kristen

Dr. med Priv.-Doz Kristen Rak

Microtubule and auditory function -an underestimated connection

Keywords: tubulin co-folding, tubulin, development, cochlea, posttranslational modifications, inner ear

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The receptor organ for hearing is the organ of Corti, located in the cochlea within the inner ear. This organ converts auditory signals into neuronal action potentials, which are transmitted to the auditory brainstem for further processing. The mature organ of Corti consists of various highly differentiated ectodermal epithelial cells, which form a mosaic-like pattern during embryonic and postnatal development [START_REF] Fritzsch | Auditory system: development, genetics, function, aging and diseases[END_REF]. At postnatal day (p)14, the sensory epithelium of mice is fully developed and animals begin to hear [START_REF] Mikaelian | Cochlear Potentials and 8 Nerve Action Potentials in Normal and Genetically Deaf Mice[END_REF]. Within the organ of Corti, the sensory hair cells are arranged in four rows: one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). Supporting cells including the inner and outer pillar cells, the inner phalangeal cell, the Deiters cells, and border cells (Hensen's cells, tectal cells, Claudius cells, Boettcher cells) contribute crucially to the stability of the sensory epithelium [START_REF] Zetes | Structure and mechanics of supporting cells in the guinea pig organ of Corti[END_REF] (Figure 1).

Microtubules form an essential part of the cytoskeleton that play key roles in cells, such as maintaining cellular structure, or intracellular transport. While the basic microtubule structure is highly conserved between different cell types and organisms, they can be composed of different tubulin isotypes, or undergo a variety of posttranslational modifications (PTMs).

Tubulin isotypes and PTMs are thought to alter the intrinsic properties of microtubule, and to control their interactions with microtubule-associated proteins (MAPs) [START_REF] Janke | The tubulin code and its role in controlling microtubule properties and functions[END_REF]. Consequently, they could also affect microtubule dynamics and functions in the cochlea.

First investigation of microtubules in the organ of Corti in gerbils found a cell-and time-specific distribution of tubulin PTMs [START_REF] Tannenbaum | Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development[END_REF]. Later, the distribution of β-tubulin isotypes in the cochlea duct was mapped during the first postnatal month [START_REF] Hallworth | Differential expression of beta tubulin isotypes in the adult gerbil cochlea[END_REF][START_REF] Saha | Age-related changes in microtubules in the guinea pig organ of Corti[END_REF]. However, the role of microtubule isotypes and PTMs in the mammalian organ of Corti, and their implications for hearing have not been investigated thoroughly. More recently, a cell-and time-specific segmentation of tubulin PTMs in murine cochlea was published [START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF].

Our review summarises the current knowledge on the distribution and the potential role of microtubules, MAPs, tubulin isotypes and PTMs in cochlear functions. By critically reviewing the potential implications of these regulators of microtubules on hearing, we aim at providing a fresh impulse to the field of microtubules and hearing. The hair cells are surrounded by different supporting cells. Deiters cells englobe the OHCs basally like a cup, while inner phalangeal cells brace the IHC. Pillar cells separate and support both the OHCs and the IHCs. Additionally, the organ of Corti is marked off medially by greater epithelial ridge cells and laterally by Hensen's, Tectal and Claudius cells.

Microtubules and their tubulin building blocks

Formation of microtubules

Microtubules are highly conserved tubular filaments with a diameter of 25 nm that are assembled from heterodimers of aand b-tubulin (Figure 2a). Several strictly controlled pathways are involved in tubulin synthesis and folding [START_REF] Cleveland | Autoregulated instability of tubulin mRNAs: a novel eukaryotic regulatory mechanism[END_REF]. In most cells, microtubules originate from the centrosome, also known as the microtubule-organising centre (MTOC), where the minus end of microtubules is anchored, while microtubules grow at the plus ends [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF][START_REF] Kellogg | The centrosome and cellular organization[END_REF] (Figure 2b). Microtubules can spontaneously switch between phases of growth and disassembly, or catastrophe, a process called "dynamic instability". The growth rate and catastrophe frequency are controlled by the hydrolysis of the GTP molecule bound to b-tubulin [START_REF] Mitchison | Dynamic instability of microtubule growth[END_REF], and by the availability of free tubulin [START_REF] Walker | Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies[END_REF]. Binding of a variety of MAPs can stabilise microtubules thereby preventing depolymerisation [START_REF] Bartolini | Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E[END_REF], while microtubule-destabilising proteins can promote depolymerisation [START_REF] Cassimeris | Regulation of microtubule-associated proteins[END_REF]. In cilia and flagella, which are involved in cell motility, microtubules assemble into axonemes, which are the molecular machines that generate ciliary beating (Figure 2c,d). In many cells, microtubules constitute polar networks (Figure 2e). These networks provide tracks for the intracellular transport of organelles and are involved in the maintenance of cell shape. During mitosis or meiosis, microtubules reorganise into a structural scaffold essential for the separation of chromosomes, known as spindles [START_REF] Behnke | A comparative study of microtubules of disk-shaped blood cells[END_REF][START_REF] Allen | Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport[END_REF][START_REF] Inoue | Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement[END_REF].

In mammalian cells, tubulin is one of the most abundant proteins [START_REF] Francis | A Trimer Consisting of the Tubulin-specific Chaperone TBCD, Regulatory GTPase ARL2, and beta-tubulin is Required for Maintaining the Microtubule Network[END_REF]. To limit the tubulin content of cells, tubulin mRNA stability is controlled by the tetratricopeptide protein 5 (TTC5), which recognises the nascent tubulin mRNA and degrades it in the presence of too high amounts of tubulin [START_REF] Lin | TTC5 mediates autoregulation of tubulin via mRNA degradation[END_REF]. Newly synthesised α-and β-tubulins are initially protected from incorrect folding by association with prefoldin [START_REF] Vainberg | Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin[END_REF], from which they are then delivered to the chaperon complex CCT (cytosolic chaperonin containing T-complex polypeptides). Correctly folded tubulin is released from CCT, however, to form a a/b-tubulin heterodimer, five more tubulin folding cofactors, TBCA, TBCB, TBCC, TBCD and TBCE are required [START_REF] Cowan | Type II chaperonins, prefoldin, and the tubulin-specific chaperones[END_REF][START_REF] Szymanski | Tubulin folding cofactors: half a dozen for a dimer[END_REF][START_REF] Gao | A cytoplasmic chaperonin that catalyzes beta-actin folding[END_REF]. The exact sequence of events involving tubulin folding cofactors has not yet been fully clarified [START_REF] Francis | A Trimer Consisting of the Tubulin-specific Chaperone TBCD, Regulatory GTPase ARL2, and beta-tubulin is Required for Maintaining the Microtubule Network[END_REF].

Tubulin heterodimers polymerise in a polar fashion to form so-called protofilaments [START_REF] Bryan | Are cytoplasmic microtubules heteropolymers?[END_REF], which by side-to-side interaction form the hollow cylinder known as the microtubule [START_REF] Erickson | Assembly of microtubules from preformed, ring-shaped protofilaments and 6-S tubulin[END_REF].

2.2.Posttranslational modifications of tubulin

Tubulin is subject to a variety of PTMs that are expected to play an important role in the generation of functionally distinct of microtubules. Tubulin PTMs can affect the properties of microtubules by either changing their intrinsic properties, or by modulating their interactions with MAPs [START_REF] Janke | The tubulin code and its role in controlling microtubule properties and functions[END_REF][START_REF] Hammond | Tubulin modifications and their cellular functions[END_REF][START_REF] Bodakuntla | Microtubule-Associated Proteins: Structuring the Cytoskeleton[END_REF]. Many of the well-characterised tubulin PTMs occur on the C-terminus of α-and β-tubulin (Figure 3), which is located on the outer surface of microtubules and thus accessible to microtubule interactors. Tubulin PTMs can also influence the stability of microtubules by affecting the speed at which they are polymerised and depolymerised [START_REF] Aillaud | Evidence for new Cterminally truncated variants of alpha-and beta-tubulins[END_REF].

First insight into the role of tubulin PTMs was gained in mitosis, the development and maintenance of neurons, as well as the function of cilia and flagella [START_REF] Janke | The tubulin code and its role in controlling microtubule properties and functions[END_REF].

Detyrosination and re-tyrosination

The C-terminal amino acid of most α-tubulins is tyrosine [START_REF] Valenzuela | Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs[END_REF]. Tyrosine can be enzymatically removed by enzymes of the vasohibin (VASH) family, which form a complex with the small vasohibin binding protein (SVBP) [START_REF] Aillaud | Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation[END_REF][START_REF] Nieuwenhuis | Vasohibins encode tubulin detyrosinating activity[END_REF] (Figure 3, blue). Detyrosinated tubulin is retyrosinated by tubulin tyrosine ligase (TTL) [START_REF] Ersfeld | Characterization of the tubulin-tyrosine ligase[END_REF]. Detyrosination occurs mainly at microtubules, while tyrosination takes place exclusively on unpolymerized α/β-tubulin heterodimers [START_REF] Hallak | Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin[END_REF][START_REF] Kumar | Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin[END_REF]. Detyrosinated tubulin can be further modified by deglutamylases that remove a glutamic acid residue, thus giving rise to ∆2-tubulin (Figure 3, light blue ∆2-tubulin).

This reaction is irreversible [START_REF] Paturle-Lafanechere | Characterization of a major brain tubulin variant which cannot be tyrosinated[END_REF][START_REF] Rudiger | The carboxy-terminal peptide of detyrosinated alpha tubulin provides a minimal system to study the substrate specificity of tubulin-tyrosine ligase[END_REF], as TTL cannot tyrosinate ∆2-tubulin [START_REF] Prota | Structural basis of tubulin tyrosination by tubulin tyrosine ligase[END_REF]. In a subsequent deglutamylation step, ∆3-tubulin is generated (Figure 3, light blue ∆3-tubulin). It is still unclear whether this reaction is reversible and which physiological effects it may cause [START_REF] Aillaud | Evidence for new Cterminally truncated variants of alpha-and beta-tubulins[END_REF][START_REF] Berezniuk | Cytosolic carboxypeptidase 1 is involved in processing alpha-and beta-tubulin[END_REF]. Tyrosinated, i.e. non-modified tubulin is predominantly found in dynamic microtubules [START_REF] Webster | Differential turnover of tyrosinated and detyrosinated microtubules[END_REF], while detyrosinated tubulin is present in rather stable microtubules [START_REF] Khawaja | Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level[END_REF]. The absence of tyrosinated tubulin prevents the depolymerisation of microtubules [START_REF] Infante | Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap[END_REF][START_REF] Peris | Motor-dependent microtubule disassembly driven by tubulin tyrosination[END_REF]. In neurons, tyrosinated microtubules are enriched in the growth cone of axons [START_REF] Witte | Microtubule stabilization specifies initial neuronal polarization[END_REF][START_REF] Baas | Individual microtubules in the axon consist of domains that differ in both composition and stability[END_REF], a region with a high degree of microtubule dynamics [START_REF] Tanaka | The role of microtubule dynamics in growth cone motility and axonal growth[END_REF], whereas axon shafts that contain long-lived, less dynamic microtubules are rich in detyrosinated and ∆2-tubulin. Acetylation of K40 on α-tubulin, located inside microtubules, is catalysed by αTAT1, and removed by either HDAC6 or SIRT2. Acetylation of β-tubulin has been reported, but is little explored. αTAT: α-tubulin N-acetyltransferase, CCP: cytosolic carboxypeptidase; HDAC6: histone deacetylase 6, SIRT2: sirtuin2; TTL: tubulin tyrosine ligase; TTLL: TTL-like.

Acetylation

Acetylation of tubulin is one of the major PTMs that is predominantly found on long-lived, often referred to as stable, microtubules [START_REF] Janke | The tubulin code: molecular components, readout mechanisms, and functions[END_REF]. The so-far best-characterised acetylation event takes place at lysine (K) 40 of α-tubulin, which lies on the luminal side of the microtubule. K40 acetylation is catalysed by α-tubulin-N-acetyltransferases 1 (αTAT1) [START_REF] Shida | The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation[END_REF][START_REF] Akella | MEC-17 is an alpha-tubulin acetyltransferase[END_REF] (Figure 3, yellow). αTAT1 has a catalytic rate lower than the lifetime of dynamic microtubules, which could be the reason why only long-lived microtubules are acetylated in cells [START_REF] Szyk | Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase[END_REF]. While tubulin acetylation preferentially occurs on polymerised microtubules [START_REF] L'hernault | Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine[END_REF][START_REF] Maruta | The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules[END_REF], deacetylation, catalysed by the histone deacetylase 6 (HDAC6, [START_REF] Hubbert | HDAC6 is a microtubule-associated deacetylase[END_REF]), or sirtuin 2 (SIRT2, [START_REF] North | The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase[END_REF]) is associated with depolymerisation of microtubules [START_REF] Black | Dynamics of alpha-tubulin deacetylation in intact neurons[END_REF]. Biophysical and structural studies revealed that K40 acetylation makes microtubules more resistant to mechanical damage [START_REF] Portran | Tubulin acetylation protects long-lived microtubules against mechanical ageing[END_REF] by facilitating the sliding of the protofilaments against each other [START_REF] Eshun-Wilson | Effects of alpha-tubulin acetylation on microtubule structure and stability[END_REF], thus promoting microtubule longevity. In the nematode C. elegans, which contains microtubules with different numbers of protofilaments, K40 acetylation is involved in the control of protofilament numbers [START_REF] Cueva | Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules[END_REF][START_REF] Topalidou | Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization[END_REF][START_REF] Renauld | Dispensability of Tubulin Acetylation for 15-protofilament Microtubule Formation in the Mammalian Cochlea[END_REF].

Another acetylation site was discovered on K252 of β-tubulin, but little is known so far on its functions [START_REF] Chu | A novel acetylation of beta-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation[END_REF].

Polyglutamylation and polyglycylation

Polyglutamylation and polyglycylation are two PTMs that have initially been discovered on tubulin [START_REF] Eddé | Posttranslational glutamylation of alpha-tubulin[END_REF][START_REF] Redeker | Structure of the polyglutamyl side chain posttranslationally added to alpha-tubulin[END_REF][START_REF] Bré | Tubulin polyglycylation: differential posttranslational modification of dynamic cytoplasmic and stable axonemal microtubules in paramecium[END_REF]. Both PTMs are linked to glutamic acids within the C-terminal tails of α-and β-tubulin, and consist in the addition of a single, or several glutamic or glycine residues, thus creating polyglutamate or polyglycine chains (Figure 3, red or green). The addition of glutamate and glycine is catalysed by enzymes of the tubulin tyrosine ligase like (TTLL) family [START_REF] Janke | Tubulin polyglutamylase enzymes are members of the TTL domain protein family[END_REF][START_REF] Rogowski | Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation[END_REF][START_REF] Wloga | TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia[END_REF][START_REF] Van Dijk | A targeted multienzyme mechanism for selective microtubule polyglutamylation[END_REF].

Polyglutamylation is reversed by deglutamylases from the cytosolic carboxypeptidase (CCP) family [START_REF] Rogowski | A family of protein-deglutamylating enzymes associated with neurodegeneration[END_REF], however deglycylating enzymes have so far not been described.

Polyglutamylation is assumed to regulate interactions between microtubules and their interacting proteins. It is found in a variety of cells and tissues; however, it is particularly enriched in neurons as well as in cilia and flagella. Recent work shows that accumulation of polyglutamylation on microtubules following inactivation CCPs causes neurodegeneration and perturbs cargo transport on neuronal microtubules [START_REF] Magiera | Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport[END_REF][START_REF] Bodakuntla | Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons[END_REF][START_REF] Bodakuntla | Tubulin polyglutamylation, a regulator of microtubule functions, can cause neurodegeneration[END_REF][START_REF] Shashi | Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration[END_REF] .

The strict localisation of glycylation to cilia and flagella suggests a particularly important functions in these organelles [START_REF] Gadadhar | The tubulin code at a glance[END_REF]. A recent study showed that loss of glycylation in mice leads to aberrant sperm swimming, which is caused by a perturbation of ordered interactions between dynein motors and microtubules in flagella [START_REF] Gadadhar | Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility[END_REF].

2.3.Tubulin isotypes

In many organisms α-and β-tubulins are encoded by multiple genes, which give rise to the expression of distinct tubulin isotypes. In humans, nine α-and nine β-tubulin isotypes have been identified [START_REF] Luduena | A hypothesis on the origin and evolution of tubulin[END_REF]. Despite their high degree of conservation, some of the β-tubulin isotypes are less conserved like neuronal β3-(TUBB3) or platelet-specific β1-tubulin (TUBB1) [START_REF] Luduena | Multiple forms of tubulin: different gene products and covalent modifications[END_REF][START_REF] Sullivan | Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes[END_REF].

The "multi-tubulin hypothesis" initially proposed that different tubulin isotypes are needed for generating functionally different microtubules [START_REF] Cleveland | The multitubulin hypothesis revisited: what have we learned?[END_REF][START_REF] Fulton | Selective synthesis and utilization of flagellar tubulin. The multitubulin hypothesis[END_REF]. This hypothesis is difficult to prove, as multiple tubulin isotypes are co-expressed in tissue and cells. However, different cell types of a tissue can have specific expression patterns of tubulin isotypes, thus indicating that isotypes might have functionally distinct roles in these cells [START_REF] Hallworth | Differential expression of beta tubulin isotypes in the adult gerbil cochlea[END_REF][START_REF] Roach | Preparation of a monoclonal antibody specific for the class I isotype of beta-tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues[END_REF][START_REF] Havercroft | Programmed expression of beta-tubulin genes during development and differentiation of the chicken[END_REF]. The demonstration that microtubules containing the β-tubulin isotype TUBB3 are more dynamic than those made of TUBB2 [START_REF] Ti | Human beta-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability[END_REF] provided a first proof of the concept that tubulin isotypes can directly affect intrinsic microtubule properties. Another way by which tubulin isotypes could influence microtubule functions are their primary sequence differences in the C-terminal tails, which could affect tubulin PTMs that take place on these tails, thereby controlling interactions between microtubules and their associated proteins [START_REF] Gadadhar | The tubulin code at a glance[END_REF].

Microtubule heterogeneity in the organ of Corti

Microtubules are prominent components of cells in the organ of Corti [START_REF] Zenner | Cytoskeletal and muscle-like elements in cochlear hair cells[END_REF]. The distribution of microtubules in the cochlea is cell-type specific and described to be relatively unchanged between humans and other so-far investigated mammalian species [START_REF] Anniko | Cytoskeletal basis for contractility of outer hair cells in the normal adult human organ of Corti: comparisons with vestibular hair cells[END_REF][START_REF] Nishizaki | Comparative cytoskeletal analyses of the inner ear in man and the squirrel monkey[END_REF]. Moreover, the cochlea is the only vertebrate tissue in which so-far microtubules with 15 protofilaments instead of the canonical 13 have been observed [START_REF] Tucker | A cell surfaceassociated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea[END_REF]. The only other described case is the nematode C. elegans, where 15-protofilament microtubules are found in the touch receptor neurons [START_REF] Chalfie | Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans[END_REF]. F-actin-free, but microtubule-rich (green) apical domain supporting the cuticular plate, with longitudinally arranged microtubules apically-basally, as well as around the nucleus.

3.1.Hair Cells

Developmental stages

During development, each hair cell possesses a kinocilium, which is a microtubule-based organelle built around a distinct microtubule axoneme and separated from the extensive microtubule network in the soma. In mice, microvilli and the kinocilium first appear at embryonic day (E) 13.5. The development, structure and function of the kinocilium is similar to other mammalian primary cilia [START_REF] Satir | Overview of structure and function of mammalian cilia[END_REF]. Only some of the microvilli surrounding the kinocilium will enlarge and become stereocilia, whereas the remaining ones will be reabsorbed to finally disappear (Figure 4a). Stereocilia grow in a defined V-shaped pattern with the kinocilium at the vertex of the "V" [START_REF] Frolenkov | Genetic insights into the morphogenesis of inner ear hair cells[END_REF][START_REF] Kelly | Shaping the mammalian auditory sensory organ by the planar cell polarity pathway[END_REF] on OHCs, while they form straight lines on IHCs (Figure 4c). In OHCs, the stereocilia are shorter than the kinocilium, whereas IHCs have kinocilia and stereocilia of similar lengths [START_REF] Li | Architecture of the mouse utricle: macular organization and hair bundle heights[END_REF]. Kinocilia spontaneously regress postnatally, and entirely disappear until p21 [START_REF] Lim | Developmental morphology of the mouse inner ear. A scanning electron microscopic observation[END_REF].

The formation of the microtubule network in the soma of hair cells starts with the first appearance of the MTOC at p0 in IHCs, and at p2 in OHCs [START_REF] Hallworth | Tubulin expression in the developing and adult gerbil organ of Corti[END_REF]. Overall concentrations of tubulin appear to play an important role in the development of hair cells. The Fibroblast Growth Factor (FGF) signalling pathway, for instance, has been demonstrated to control the expression of cytoskeletal components, including tubulin and regulators of microtubule dynamics. Mice lacking FGF receptor (Fgfr3-KO mice) show incorrect development and differentiation of OHCs and supporting cells [START_REF] Szarama | Fibroblast growth factor receptor 3 regulates microtubule formation and cell surface mechanical properties in the developing organ of Corti[END_REF]. In the adult cochlea of wildtype mice, a general decrease in tubulin concentration at the apical and the medio-basal domains has been observed [START_REF] Vago | Distribution of actin and tubulin in outer hair cells isolated from developing rat cochlea: a quantitative study[END_REF]. This cell type-specific reduction in tubulin in hair cells occurs at a late stage of development [START_REF] Jensen-Smith | Cell type-specific reduction of β tubulin isotypes synthesized in the developing gerbil organ of Corti[END_REF].

Adult stage

In the adult hair cells, microtubules are found mainly in the supranuclear and apical parts of the cells [START_REF] Furness | Organization of microtubules in cochlear hair cells[END_REF]. They are running parallel to the main axis of the cell body, which is directly connected to the cuticular plate in the actin-free domains [START_REF] Jorgensen | Microtubules and laminated structures in inner ear hair cells[END_REF][START_REF] Raphael | F-actin, tubulin and spectrin in the organ of Corti: comparative distribution in different cell types and mammalian species[END_REF] (Figure 4b). The cytoplasm contains less microtubules compared to the apical domain [START_REF] Jorgensen | Microtubules and laminated structures in inner ear hair cells[END_REF]. In inner hair cells, microtubules are also found at the lateral membrane of the cells [START_REF] Furness | Organization of microtubules in cochlear hair cells[END_REF][START_REF] Raphael | F-actin, tubulin and spectrin in the organ of Corti: comparative distribution in different cell types and mammalian species[END_REF][START_REF] Steyger | Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure[END_REF]. From the apical part of the cell microtubules stretch down into the hair cell [START_REF] Slepecky | Actin-binding and microtubule-associated proteins in the organ of Corti[END_REF]. There are quantitatively less microtubules in the basal part of the hair cell [START_REF] Raphael | F-actin, tubulin and spectrin in the organ of Corti: comparative distribution in different cell types and mammalian species[END_REF], where some of them connect to neuronal synapses [START_REF] Jorgensen | Microtubules and laminated structures in inner ear hair cells[END_REF].

Past studies used specific antibodies to visualise the presence of different tubulin isotypes and PTMs in the hair cells. OHCs contained ßI-(TUBB5) and ßIV-tubulin (TUBB4) isotypes, while IHCs were positive for ßI-(TUBB5) and ßII-tubulin (TUBB2) isotypes (both the original and the new nomenclatures are given) [START_REF] Hallworth | Differential expression of beta tubulin isotypes in the adult gerbil cochlea[END_REF][START_REF] Jensen-Smith | Cell type-specific reduction of β tubulin isotypes synthesized in the developing gerbil organ of Corti[END_REF]. PTMs appear to be less enriched, as tyrosination (i.e. non-detyrosinated a-tubulin) was the main form found in the sensory hair cells [START_REF] Tannenbaum | Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development[END_REF][START_REF] Slepecky | Actin-binding and microtubule-associated proteins in the organ of Corti[END_REF][START_REF] Pack | Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients[END_REF][START_REF] Slepecky | Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea[END_REF][START_REF] Ladrech | Changes in MAP2 and tyrosinated alpha-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat[END_REF] (Figure 5a). Detyrosination and polyglutamylation, form only a small component of cytoplasmic microtubules located below the cuticular plate, while acetylated form is more prominent [START_REF] Slepecky | Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea[END_REF]. The distribution of tyrosinated tubulin within the sensory cells does not change as animals age, but an age-related decrease in the number and the length of microtubules as well as an increase of soluble tubulin has been reported [START_REF] Saha | Age-related changes in microtubules in the guinea pig organ of Corti[END_REF].

Supporting cells

During development of supporting cells, the microtubule cytoskeleton occurs first at the cell apex, from where it polymerises basally [START_REF] Ito | Cytological changes related to maturation of the organ of Corti and opening of Corti's tunnel[END_REF]. Finally, microtubules are arranged longitudinally to the axis of the cell. The main function of microtubules in supporting cells is to stabilise the sensory hair cells and to provide resilience to mechanical forces from the basilar and tectorial membrane during the travelling of sound waves in the inner ear.

Pillar cells

Detailed immunohistochemical analyses revealed that microtubules are present in the foot, shaft and apical plate of the outer pillar cells [START_REF] Flock | Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells[END_REF]. Microtubules in apical plates and the narrow phalangeal processes are densely organised and contain ßI-(TUBB5), ßII-(TUBB2), ßIV-(TUBB4) and ßV-tubulin (TUBB6) [START_REF] Jensen-Smith | Cell type-specific reduction of β tubulin isotypes synthesized in the developing gerbil organ of Corti[END_REF][START_REF] Renauld | Spatio-temporal dynamics of betatubulin isotypes during the development of the sensory auditory organ in rat[END_REF] (Figure 6). Tubulin PTMs emerge on microtubules starting from the cell apex toward the base, i.e. in the same direction in which microtubules elongate. The appearance of acetylation is rapidly followed by tubulin detyrosination between p2 and p4 [START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF][START_REF] Renauld | Dispensability of Tubulin Acetylation for 15-protofilament Microtubule Formation in the Mammalian Cochlea[END_REF][START_REF] Liu | Expression of trans-membrane serine protease 3 (TMPRSS3) in the human organ of Corti[END_REF].

In inner pillar cells, microtubule bundles are organised by MTOCs, which connect about 3,000 microtubules each [START_REF] Henderson | Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning[END_REF]. The intensity of α-tubulin labelling in these cells is not altered with increasing age [START_REF] Tucker | A cell surfaceassociated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea[END_REF][START_REF] Henderson | Reorganization of the centrosome and associated microtubules during the morphogenesis of a mouse cochlear epithelial cell[END_REF]. In contrast, the extent of labelling across the outer pillar cells is reduced in higher ages, suggesting the presence of fewer, but still tightly packed microtubules [START_REF] Saha | Age-related changes in microtubules in the guinea pig organ of Corti[END_REF].

Deiters cells

Deiters cells contain large numbers of microtubules [START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF][START_REF] Flock | Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells[END_REF]. At the level of the cup-like structure of these cells, microtubules support the basal part of OHCs to enhance their stability [START_REF] Spicer | Cytologic structures unique to Deiters cells of the cochlea[END_REF]. At the base, near the basilar membrane, microtubules are abundant [START_REF] Parsa | Deiters cells tread a narrow path--the Deiters cellsbasilar membrane junction[END_REF] and the stalk ramifies into smaller microtubule bundles in apical cytosol [START_REF] Spicer | Cytologic structures unique to Deiters cells of the cochlea[END_REF]. A recent study showed that Gas9, a MAP, has a microtubule-stabilising function at the apical region of Deiters cells [START_REF] Chen | Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing[END_REF].

Similar to what has been observed in pillar cells, microtubules in Deiters cells are acetylated, while detyrosination appears one or several days later. At onset of cochlear function, microtubule detyrosination and acetylation have achieved their adult-like patterns, whereas polyglutamylated tubulin reached its highest levels only at about 6 weeks after birth [START_REF] Tannenbaum | Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development[END_REF][START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF]. The observation of an age-related decrease in labelling for tubulin and detyrosinated tubulin [START_REF] Saha | Age-related changes in microtubules in the guinea pig organ of Corti[END_REF] might indicate a loss of microtubules in these cells. Tubulin isotypes detected in Deiters cells are βII (TUBB2) and βIV (TUBB4) [START_REF] Renauld | Spatio-temporal dynamics of betatubulin isotypes during the development of the sensory auditory organ in rat[END_REF] (Figure 6).

Other supporting cells

During development, a transient structure known as the Kölliker's organ presents an assembly of epithelial cells in immature cochlea. Tyrosinated and acetylated microtubules are found in the area around the basal part of the Kölliker's organ, as well as in tectal and Hensen's cells [START_REF] Tannenbaum | Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development[END_REF]. The high levels of tyrosinated tubulin in cells of the Kölliker's organ, as well as the expression of tubulin specific co-folding factors in Kölliker's organ and lateral border cells [START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF], are both signs of high tubulin turnover in this developmentally transient part of the organ of Corti. Polyglycylation is found in inner border cell as wells as inner and outer phalangeal cells, where also (f) polyglutamylation can be detected.

3.3.Vestibular organs

Apart from the cochlea, microtubules were also investigated in the vestibular part of the inner ear, where the functional organ is called maculae organ that contributes to the sense of balance.

Microtubules are abundant in the nuclear regions of type-I hair cells, but sparse in type-II hair cells of the vestibular epithelium [START_REF] Kikuchi | Microtubule subunits of guinea pig vestibular epithelial cells[END_REF]. The expression of tubulin isotypes varies in the vestibular system. Sensory hair cells selectively express ßIV-(TUBB4) and ßI-tubulin (TUBB5), while supporting cells express ßI-(TUBB5), ßII-(TUBB2), and ßIV-tubulin (TUBB4). Neuronal fibres show specific expression of ßI, ßII, and ßIII-tubulin (TUBB3) [START_REF] Perry | Selective expression of beta tubulin isotypes in gerbil vestibular sensory epithelia and neurons[END_REF].

Supporting and hair cells of the maculae assemble cytoskeletal bundles of microtubules and actin filaments, which are mostly arranged longitudinally in the cells. The observation that these bundles are arranged around centrioles indicated they have served as nucleation centres of microtubules [START_REF] Heywood | Distribution of microtubules and microfilaments in developing vestibular sensory epithelium of mouse otocysts grown in vitro[END_REF]. Nevertheless, the number of microtubules in vestibular supporting cells is far smaller than that of cochlear supporting cells. It is likely that a smaller number of microtubules is sufficient to ensure the stability of vestibular supporting cells, given that the vestibular sensory epithelium is specialised for detection of lower frequency vibrations that are less energy-intense [START_REF] Kikuchi | Microtubule subunits of guinea pig vestibular epithelial cells[END_REF].

Distribution and roles of Microtubule-Associated Proteins (MAPs) in the organ of

Corti

Microtubules interact with a wide variety of MAPs, which play important roles regulating the architecture of the cytoskeleton by controlling the dynamics of microtubules [START_REF] Bodakuntla | Microtubule-Associated Proteins: Structuring the Cytoskeleton[END_REF]. The role of MAPs has most extensively been investigated in neurogenesis, neuronal plasticity and neuronal homeostasis and degeneration [START_REF] Penazzi | Microtubule Dynamics in Neuronal Development, Plasticity, and Neurodegeneration[END_REF], but less is known on their roles in the inner ear.

In the cochlea, the expression of MAP1A was detected in the supporting cells of the cristae, maculae, nerve fibres and in the apical region adjacent to the sensory hair cells [START_REF] Takumi | Microtubule associated protein (MAP1A) mRNA was up-regulated by hypergravity in the rat inner ear[END_REF] (Figure 6a). During neurite outgrowth of auditory neurons, MAP1B (earlier known as MAP5) is highly expressed in cochleovestibular ganglions, but decreases at the end of this developmental period. MAP1B could thus be involved in controlling specific microtubule functions during neuronal development [START_REF] Jose | Expression of the cytoskeletal protein MAP5 and its regulation by neurotrophin 3 (NT3) in the inner ear sensory neurons[END_REF] (Figure 6b). In a recent study, spiral ganglion neurons of mice carrying a mutation in MAP1B mice exhibited a deficit in MAP1B phosphorylation, as well as perturbed microtubule dynamics, which resulted in a sensorineural hearing loss due to dysfunction of spiral ganglions [START_REF] Cui | Mutations of MAP1B encoding a microtubuleassociated phosphoprotein cause sensorineural hearing loss[END_REF]. MAP2 can already be detected at E16 in the cell soma and the axon of the spiral ganglion.

Within the first postnatal week, MAP2 is also expressed in dendrites and synapses [START_REF] Hafidi | Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear[END_REF] (Figure 6c). After treatment of the organ of Corti with the ototoxin amikacin, an aminoglycoside inducing hearing loss, an increase in the expression of the embryonic splice isoform of MAP2, MAP2C, was observed. Together with an increase of tyrosinated tubulin, this observation suggested a limited repair process of the sensory hair cells, which had previously been considered to have lost the ability to recover once they are mature [START_REF] Ladrech | Changes in MAP2 and tyrosinated alpha-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat[END_REF]. Moreover, it indicates a potential role of microtubules in the regeneration process of the organ of Corti.

The neuronal MAP tau plays an important role in healthy neurons and its dysfunction leads to neurodegenerative disorders such as Alzheimer's disease [START_REF] Goedert | Tau filaments in neurodegenerative diseases[END_REF]. Tau was detected in the nerve fibres below both inner and outer hair cells and in the osseous spiral lamina [START_REF] Slepecky | Actin-binding and microtubule-associated proteins in the organ of Corti[END_REF]. By contrast, tau could not be found in the sensory cells in the organ of Corti, while other neuronal MAPs, such as MAP1A, MAP1B and MAP2, were detected.

Another MAP, MAP1S (also known as MAP8), interacts with Prestin [START_REF] Zheng | Prestin is the motor protein of cochlear outer hair cells[END_REF], a protein of the family of transmembrane anion transporters that mediate electroneutral exchange of chloride and carbonate across the plasma membrane of mammalian cells [START_REF] Bai | Prestin's Anion Transport and Voltage-Sensing Capabilities Are Independent[END_REF][START_REF] Song | Conformational State-Dependent Anion Binding in Prestin: Evidence for Allosteric Modulation[END_REF]. Substitution of chloride by sulphate in the binding pocket of Prestin leads to a conformational rearrangement of two structural states of this protein [START_REF] Bavi | The conformational cycle of prestin underlies outer-hair cell electromotility[END_REF]. This molecular motion is several orders of magnitude faster than what has been observed for other motor proteins [127], and exert lateral forces that directly influence the physical state of the plasma membrane. The process mechanically contracts and elongates OHCs, a process known as electromotility. Prestin is the driving force behind the somatic motor of the cochlear amplifier, which in mammals increases sensitivity to incoming sound wave frequencies and thus amplifies the signal [START_REF] Oliver | Intracellular Anions as the Voltage Sensor of Prestin, the Outer Hair Cell Motor Protein[END_REF]. MAP1S is implicated in delivering newly synthesised Prestin to the cell surface, thereby supporting electromotility and increasing the voltage sensitivity, particularly in high-frequency-sensing hair cells [START_REF] Bai | Prestin surface expression and activity are augmented by interaction with MAP1S, a microtubule-associated protein[END_REF].

Other microtubule-interacting proteins

Beside the "classical" MAPs discussed above, there are several other proteins interacting with microtubules that have been shown to play specific roles in hearing.

Marshalin, or calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a microtubule minus-end binding protein, is abundantly expressed during cochlear development in both sensory hair cells and supporting cells. Marshalin is prominently expressed at the cuticular plate of hair cells at basal bodies, Deiters and pillar cells [START_REF] Zheng | Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti[END_REF]. CAMSAP3-deficient mice showed disturbed audio records [START_REF] Robinson | CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia[END_REF], however the functional roles of Marshalin in the organ of Corti remain yet to be identified. Spectrins are sub-membranous cytoskeletal structures found in the lateral plasma membrane of cochlear OHCs, where they are involved in maintaining the specialised cell shape and the electromotility of the these cells [START_REF] Mahendrasingam | Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti[END_REF]. The unconventional spectrin βV is essential in this process as it regulates membrane trafficking at the lateral wall of outer hair cells [START_REF] Cortese | Spectrin βV adaptive mutations and changes in subcellular location correlate with emergence of hair cell electromotility in mammalians[END_REF], most likely via actin-and microtubule-based motors [START_REF] Papal | The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route[END_REF].

Septins are a family of GTP-binding proteins known for their essential roles in morphogenesis, and physiological functions like regulation of microtubule stability, trafficking, and planar cell polarity [START_REF] Yoshida | Localization of septin proteins in the mouse cochlea[END_REF][START_REF] Spiliotis | Cellular functions of actin-and microtubule-associated septins[END_REF]. In the fully developed organ of Corti, Septins are expressed in supporting cells as well as sensory cells [START_REF] Yoshida | Localization of septin proteins in the mouse cochlea[END_REF]. During development, only Septin7 is expressed, which suggests a specific role in morphogenesis [START_REF] Torii | Septin7 regulates inner ear formation at an early developmental stage[END_REF].

During embryonic development, the planar polarity of the apical part of auditory hair cells and the uniform V-shape orientation of the stereocilia bundles are regulated by the WNT-signalling pathway. The microtubule-based kinesin motor Kif3a has been identified as a key component of the hair cell-intrinsic polarity machinery. Kif3a regulates the activity of p21-activated kinases, which in turn controls the positioning of the basal bodies in hair cells [START_REF] Sipe | Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms[END_REF]. Basal bodies are centrioles similar to those found in centrosomes, which are anchored to the plasma membrane to assemble the ciliary axonemes. They thus define the position of the kinocilium, and thereby cell polarity. Another protein, Lis 1, an interactor of the microtubule-based motor dynein [START_REF] Huang | Lis1 acts as a "clutch" between the ATPase and microtubule-binding domains of the dynein motor[END_REF], has been shown to regulate p21-activated kinase Lis 1 mediates planar polarity of hair cells by regulation of cytoplasmic dynein, which affects microtubule organisation, as well as the positioning of Golgi complex and mitochondria. Strikingly, loss of Lis1 disrupts the anchoring of basal bodies, and disrupts the normal V-shape of hair bundles [START_REF] Sipe | Lis1 mediates planar polarity of auditory hair cells through regulation of microtubule organization[END_REF].

The morphology of the mechanosensitive hair bundle requires seamless integration of the actin and microtubule networks. The actin-microtubule crosslinking factor MACF1 (also known as Acf7) bridges these two cytoskeletal networks by specific protein domains: The N-terminal domains facilitate direct interaction with F-actin, while microtubule binding is mediated by the GAS2-related protein domain near the C-terminus. Both domains are connected via a long stretch of spectrin repeats, which are believed to provide an elastic connection between actin and microtubules [START_REF] Suozzi | Spectraplakins: master orchestrators of cytoskeletal dynamics[END_REF]. In vestibular and cochlear hair cells in mice, MACF1 encircles the basal body of the kinocilium. During hair-cell development and regeneration, MACF1 precedes the hair bundle and the cuticular plate at the apical surface of the presumptive hair cells [START_REF] Antonellis | ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin[END_REF].

Despite the predicted key roles of MACF1 in coordinating cytoskeletal crosstalk in hair cells, its loss in the inner ear of mice did not impede hearing [START_REF] Gilbert | Actin Crosslinking Family Protein 7 Deficiency Does Not Impair Hearing in Young Mice[END_REF]. It remains to be established whether another protein of the MACF family, MACF2 or Dystonin, which is also expressed in hair cells [START_REF] Leonova | Expression of the mouse Macf2 gene during inner ear development[END_REF], could compensate for the lack of MACF1.

Physiological functions of microtubules in the mammalian Cochlea

Sensory cells in the organ of Corti contain dynamic microtubules that are enriched in tyrosinated, i.e. non-modified tubulin [START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF][START_REF] Slepecky | Actin-binding and microtubule-associated proteins in the organ of Corti[END_REF][START_REF] Bane | Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications[END_REF][START_REF] Westermann | Post-translational modifications regulate microtubule function[END_REF]. By contrast, non-sensory supporting cells contain microtubules marked with several PTMs indicating that they might be more stable, especially acetylated tubulin [START_REF] Tannenbaum | Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development[END_REF][START_REF] Juergens | Spatio-temporal distribution of tubulin-binding cofactors and posttranslational modifications of tubulin in the cochlea of mice[END_REF][START_REF] Slepecky | Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea[END_REF]. This difference is likely to reflect functional diversity of microtubules in these two cell types. Supporting cells, which contain rather stable microtubules are essential for the mechanic stability of the organ of Corti. In contrast, dynamic microtubules in sensory cells might have roles in active functions such as protein transport, or, in the case of the hair cells, the elongation of the cell [START_REF] Bane | Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications[END_REF]. For example, the process of hair-cell elongation that results in the characteristic tubular shape of outer hair cells depends on the protein Prestin [START_REF] Zheng | Prestin is the motor protein of cochlear outer hair cells[END_REF], and its modulation by MAP1S [START_REF] Bavi | The conformational cycle of prestin underlies outer-hair cell electromotility[END_REF][START_REF] Bai | Prestin surface expression and activity are augmented by interaction with MAP1S, a microtubule-associated protein[END_REF] as described above.

The uniform orientation of hair bundles on the apical pole of auditory hair cells, which is required for the perception of sound, depends on epithelial planar cell polarity. The type of microtubule organisation required for establishing cell polarity is dependent on the positioning of the basal body. The basal body connects microtubules to the plasma membrane, where they define cell shape and polarity [START_REF] Li | Beyond polymer polarity: how the cytoskeleton builds a polarized cell[END_REF][START_REF] Bornens | Organelle positioning and cell polarity[END_REF]. Abnormal planar cell polarity, caused by disorganisation of microtubules at the plasma membrane, resulted in a mispositioning of basal body and kinocilium, as well as in disturbances in hair-bundle orientation and morphology [START_REF] Sipe | Regulation of planer cell polarity in the mammalian auditory sensory epithelium by microtubule-mediated processes[END_REF][START_REF] Kirjavainen | The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea[END_REF].

The presence of posttranslationally modified microtubules in supporting cells strongly suggests that these microtubules are stable, or long-lived, and could thus contribute to the structural support of the sensory organ of Corti [START_REF] Slepecky | Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea[END_REF]. Indeed, bundled microtubules generated by distinct MTOCs generate dense networks that connect to cell-cell junctions as well as to the basilar membrane [START_REF] Henderson | Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning[END_REF]. The establishment of these bundled microtubule arrays also depends on the adenomatous polyposis coli (APC) protein, a microtubule interactor involved in the WNTsignalling pathway [START_REF] Mogensen | The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells[END_REF].

Animal models

Several transgenic animal models were used to investigate how microtubule organisation affects the functions of the sensory epithelium. In addition, drugs causing ototoxicity were used to study the degeneration of cells of the cochlear organ of Corti. Here we present a selection of relevant study in animal models that have provided insights into the role of microtubules in the cochlea.

Ototoxicity can be induced by drugs perturbing the cytoskeleton. Paclitaxel, a widely used chemotherapeutic agent for treatment of neoplastic cancers, is a microtubule-stabilising agent that entirely supresses microtubule dynamics, which leads to arrest of cell proliferation and perturbation of other microtubule-based intracellular processes. In the inner ear, paclitaxel causes the degeneration of the sensory hair cells, auditory nerve fibres and spiral ganglia, as demonstrated in rats and guinea pigs [START_REF] Ladrech | Changes in MAP2 and tyrosinated alpha-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat[END_REF][START_REF] Takumida | The effect of gentamicin on cytoskeletons in the vestibular sensory cells: a high-resolution scanning electron microscopic investigation[END_REF][START_REF] Karlsson | Ultrastructural changes in the outer hair cells of the guinea pig cochlea after exposure to quinine[END_REF][START_REF] Dong | Ototoxicity of paclitaxel in rat cochlear organotypic cultures[END_REF], thus underpinning the essential roles of microtubules in these different cell types.

The progressive motor neuronopathy (pmn) mouse is a model for a fast-developing human motor neuron disorder. First manifestations of motor neuropathy in homozygous pmn mice become apparent at the end of the third post-natal week, and mice die up to six weeks after birth from respiratory paralysis. The disease starts at the motor endplates, leads to axonal loss and finally to apoptosis of the corresponding neuronal cell bodies [START_REF] Schmalbruch | A new mouse mutant with progressive motor neuronopathy[END_REF]. The gene defect in pmn mice was mapped to a missense mutation in the tubulin-folding cofactor E (TBCE) [START_REF] Bommel | Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease[END_REF][START_REF] Martin | A missense mutation in Tbce causes progressive motor neuronopathy in mice[END_REF], one of five tubulin-specific co-folding factors essential for the generation of functional tubulin in cells [START_REF] Tian | Pathway leading to correctly folded beta-tubulin[END_REF]. Pmn mice show axonal degeneration due to microtubule loss [START_REF] Martin | A missense mutation in Tbce causes progressive motor neuronopathy in mice[END_REF], and impairment of axonal outgrowth in cultured primary neurons [START_REF] Bommel | Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease[END_REF]. First studies regarding the auditory development of the pmn mice revealed a rapid hearing loss that starts at postnatal day 21. While pmn mice show a normal morphology of the cochlea and the spiral ganglion [START_REF] Volkenstein | Auditory development in progressive motor neuronopathy mouse mutants[END_REF], they display a reduction of microtubule numbers and disturbed arrangement of microtubule structures in the auditory nerve, as well as a progressive loss of outer hair cells due to apoptosis, which is accompanied by hearing loss [START_REF] Rak | Mutation of the TBCE gene causes disturbance of microtubules in the auditory nerve and cochlear outer hair cell degeneration accompanied by progressive hearing loss in the pmn/pmn mouse[END_REF].

The tubby mouse is another animal model with a spontaneous mutation causing sensorineural hearing loss. The gene muted in these mice, tub, encodes a protein that is part of a larger family of tubby-like proteins that are involved in ciliary trafficking [START_REF] Hong | Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking[END_REF]. Homozygote tubby mice display a loss of OHCs at the extreme cochlear base at 1 month, which progresses to the cochlear apex at around 6 months after birth. Interestingly, the spiral ganglia are degenerated only locally, in their basal turn, by 6 months [START_REF] Ohlemiller | Progression of cochlear and retinal degeneration in the tubby (rd5) mouse[END_REF]. Nevertheless, the hearing loss of tubby mouse is already apparent at 3 weeks after birth, when OHC are still present. A new study described a loss of connection between stereocilia and the tectorial membrane in tubby mouse, which leads to OHC dysfunction [START_REF] Han | Distinct roles of stereociliary links in the nonlinear sound processing and noise resistance of cochlear outer hair cells[END_REF]. Strikingly, the hearing loss of tubby mice was selectively neutralised by alleles of the MAP1A gene carrying several mutations [START_REF] Ikeda | Microtubule-associated protein 1A is a modifier of tubby hearing (moth1)[END_REF]. Given that MAP1A is essential for synaptic functions in neurons [START_REF] Takei | Defects in Synaptic Plasticity, Reduced NMDA-Receptor Transport, and Instability of Postsynaptic Density Proteins in Mice Lacking Microtubule-Associated Protein 1A[END_REF], it is supposed that the tubbyinduced hearing loss could be associated with perturbed synaptic function in neuronal cells.

CYLD is a tumour suppressor that has been characterised as microtubule-binding protein involved in the regulation of various cellular processes, such as cell division and ciliogenesis through deubiquitylation [START_REF] Yang | CYLD -a deubiquitylase that acts to fine-tune microtubule properties and functions[END_REF]. CYLD can be widely detected in mouse cochlear tissues and in the neuronal cell lineage. Knocking down CYLD in the murine cochlea leads to a significant reduction in the length and proportion of neurite outgrowth, which in turn causes a mild hearing impairment [START_REF] Yang | CYLD deficiency causes auditory neuropathy due to reduced neurite outgrowth[END_REF].

The protein ccdc88c (also known as Daple) is a scaffold protein that interacts with dishevelled (Dvl), a planar cell polarity protein that controls microtubule dynamics via the WNT-signalling pathway [START_REF] Ciani | A divergent canonical WNTsignaling pathway regulates microtubule dynamics: dishevelled signals locally to stabilize microtubules[END_REF]. Daple has been reported to regulate several biological activities through WNTsignalling [START_REF] Takagishi | Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus[END_REF]. In the ear, Daple has a fundamental role in the establishment of hair-cell polarity. Daple-KO mice show cell-intrinsic polarity defects with mis-positioned kinocilia and severely perturbed hair bundle patterning [START_REF] Siletti | Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles[END_REF]. Consequently, daple-KO mice exhibit hearing impairment that is particularly pronounced at high frequencies. Strikingly, the phenotypes observed in daple-KO mice could be mimicked by treatment with nocodazole, a tubulin polymerisation inhibitor that leads to microtubule loss in cells [START_REF] Ozono | Daple deficiency causes hearing loss in adult mice by inducing defects in cochlear stereocilia and apical microtubules[END_REF].

Human deafness related to microtubule dysfunctions

To date, only a small number of genetic diseases caused by mutation in genes linked to the microtubule cytoskeleton are known to lead to human deafness.

Leber congenital amaurosis (LCA) is a neurodegenerative disease of photoreceptor cells that causes blindness within the first year of life. Strikingly, a cohort of LCA patients that also carry heterozygous mutation of TUBB4B gene also develop early hearing loss. The causative role of the TUBB4B mutation was confirmed in a mouse model mimicking this disease. Observations on fibroblasts from these mice suggested that altered microtubule dynamics could be involved in the development of the pathology [START_REF] Luscan | Mutations in TUBB4B Cause a Distinctive Sensorineural Disease[END_REF].

Meniere's disease is a disorder of the inner ear characterised by episodes of vertigo, tinnitus and hearing loss. The cause of the disease is still unclear, but several hypotheses, including autoimmune reactions are currently considered. Surprisingly, about half of the patients appear to have antibodies against β-tubulin in the inner ear [START_REF] Yoo | β-Tubulin as an autoantigen for autoimmune inner ear disease[END_REF][START_REF] Yoo | Presence of Autoantibodies in the Sera of Meniere's Disease[END_REF]. How β-tubulin antibodies are involved in the development of Meniere's disease is still not understood. To investigate whether these antibodies could be causative for this pathology, mice were immunised to β-tubulin in the inner ear. These mice developed substantial hearing loss associated with degeneration of the organ of Corti and spiral ganglion cells [START_REF] Zhou | Experimental autoimmune hearing loss is exacerbated in IL-10-deficient mice and reversed by IL-10 gene transfer[END_REF], however, no mechanistic details of this pathological process have so-far been described.

Mutations in MAPs have also been identified in human patients with hearing loss.

Heterozygous mutations with autosomal dominant inheritance in MAP1B were linked to nonsyndromic sensorineural hearing loss. As shown in mouse models, a monoallelic mutation in this gene caused spiral ganglion cells degeneration. As some of the MAP1B mutations were found on conserved phosphorylation sites, aberrant phosphorylation causing altered microtubule interactions of MAP1B was proposed as one possible disease mechanism [START_REF] Cui | Mutations of MAP1B encoding a microtubuleassociated phosphoprotein cause sensorineural hearing loss[END_REF].

Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit recessive non-syndromic hearing loss accompanied by disorganisation and destabilisation of microtubule bundles in supporting cells. Gas2 mutant mice showed a reduced stiffness of supporting cells, which in turn altered cochlear micromechanics [START_REF] Chen | Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing[END_REF].

Another autosomal recessive non-syndromic hearing impairment, known as DFNB66, caused by a point mutation in DCDC2, a member of doublecortin-like domain containing protein superfamily [START_REF] Grati | A missense mutation in DCDC2 causes human recessive deafness DFNB66, likely by interfering with sensory hair cell and supporting cell cilia length regulation[END_REF]. DCDC2 contains a doublecortin domain known to bind microtubules [START_REF] Massinen | Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons[END_REF].

While the underlying mechanisms of this disease are still unknown, initial analyses of the cochlea showed that DCDC2 accumulates at the tip of kinocilia of sensory hair cells. This potentially links DCDC2 to ciliary assembly and function, suggesting that mutations could lead to kinocilium anomalies that could alter cell polarity, which has previously been shown to affect hearing [START_REF] Sipe | Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms[END_REF][START_REF] Massinen | Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons[END_REF].

Conclusion

Here we compiled the current knowledge on the roles of microtubules, tubulin diversity and distribution, and interactions with microtubule-interacting proteins in the inner ear. Our aim was to highlight the recently emerging importance of the microtubule cytoskeleton on the function of the cochlea, and how pathological changes in components of the microtubule cytoskeleton can cause auditive dysfunctions. While so far much of the published evidence is circumstantial, potential mechanisms and function of the identified molecular players in the sensory epithelium of the cochlea could be inferred from their better-known roles in the central and peripheral nervous systems. Given that most of the knowledge on the role of microtubules in hearing has been accumulated in recent years, we are confident that the topic will come into greater focus in the field of auditory research.

Figure 1 :

 1 Figure 1: Schematic view of sensory epithelium of the cochlea. The cochlea is separated in three different regions. Scala vestibuli and Scala tympani are filled with endolymph, whereas Scala media consist of perilymph and includes the organ of Corti. The organ of Corti is a specialised sensory epithelium that allows for the transduction of mechanical sound vibrations into electrical neural signals. The organ of Corti itself is placed on the basilar membrane, a highly specialised basement membrane, and is limited by the tectorial membrane. The organ of Corti consists of one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs).

Figure 2 :

 2 Figure 2: Microtubule architecture and cellular functions. (a) Microtubules dynamically assemble from dimers consisting of α-and β-tubulin subunits. They have a diameter of 25 nm. (b) Microtubules cycle between periods of growth and depolymerization, a property known as "dynamic instability". The transition from growth to depolymerisation is called catastrophe, while transition from depolymerisation to growth is called rescue. Microtubule dynamics is characterised by three parameters: rates of polymerisation and depolymerisation, and frequency of catastrophe. Microtubule minus ends (-End) are anchored by the microtubule-organising centre (MTOC), from which microtubules grow toward their plus ends (+End). Among their many cellular functions, microtubules drive cell motility via cilia, for example (c) by multiple motile cilia of the unicellular organism Tetrahymena thermophila, or (d) by a single sperm

Figure 3 :

 3 Figure 3: Posttranslational modifications of tubulin and their enzymes. Schematic representation of a microtubule, its PTMs, and the modifying enzymes. Tubulins are highly structured proteins, forming 'tubulin bodies' that for simplification are here represented as balls. The unstructured C-terminal tails of α-and β-tubulin are represented as amino acid sequences in grey (α-tubulin: murine α1A-tubulin, residues 441-451; β-tubulin: murine β2Btubulin, residues 432-445). Both, α-and β-tubulin are modified by polyglutamylation and polyglycylation at glutamate residues within the primary peptide chain of the protein, the socalled branching points. Both PTMs are catalysed by enzymes from TTLL family, and polyglutamylation is removed by enzymes from the CCP family. Detyrosination enzymatically removes the C-terminal tyrosine residue of α-tubulin with enzymes from the VASH family, which can be re-added by TTL. Detyrosinated tubulin can be further processed by removal of

Figure 4 :

 4 Figure 4: Overview of the development and arrangement of microtubules in sensory hair cells. (a) A single, microtubule-based kinocilium is surrounded by an asymmetric assembly of actin-based (red) microvilli. The kinocilium is thought to provide directional sensitivity. As the number of microvilli increases, the kinocilium retracts into the cuticular plate. Microvilli further develop into stereocilia. Vertebrates are unique for the highly polarised, organ-pipe-like arrangement of stereocilia. The stereocilia are attached to each other at their tips, and form either (b) a "V"-shape on mature OHCs, or straight lines on IHCs. (c) A mature hair cell has a

Figure 5 :

 5 Figure 5: Distribution of tubulin PTMs in the organ of Corti. Schematic overview of PTMs of microtubules, in the sensory epithelium of the cochlea. (a) Unmodified tyrosinated tubulin is present in the sensory hair cells and supporting cells. (b) Detyrosination can be found in Deiters cells and pillar cells. (c) Δ2-tubulin is found in inner and outer phalangeal cells and pillar cells. (d) Acetylated microtubules are present in Deiters and pillar cells. (e)

Figure 6 :

 6 Figure 6: Distribution of MAPs, β-tubulin isotypes, and tubulin specific co-folding factors in the organ of Corti. Microtubule-associated proteins (MAPs): (a) MAP1A is only detectable in the IHC; (b) MAP1B is restricted to spiral ganglions; (c) MAP2 is present in sensory hair cells and spiral ganglion nerves. Tubulin isotypes: (d) βI-tubulin (TUBB5) is found in OHCs,

Acknowledgment

The research of LB and KR is funded by DFG (RA2819/2-1). CJ and IJ are supported by ANR-10-IDEX-0001-02, the LabEx Cell'n'Scale ANR-11-LBX-0038, and the CEFIPRA (5703-1) award. We would like to thanks S. Bodakuntla (NIH, Bethesda, USA) for insightful discussions,

as well as M.M. Magiera and J. Davey for the unique opportunity to include our manuscript in this book.


Sylvain Lemettre

Clément Bessouet

Charlotte Kutyla

Alain Bosseboeuf

Philippe Coste

Thierry Sauvage

Hélène Lecocq

Olivier Wendling

Aurelien Bellamy

Johan Moulin

Integration of Zr-V getter films in MEMS packaging

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Integration of Zr-V getter films in MEMS packaging

Sylvain Lemettre 1 , Clément Bessouet 1 , Charlotte Kutyla 1 , Alain Bosseboeuf 1 , Philippe Coste 1 , Thierry Sauvage 2 , Hélène Lecoq 2 , Olivier Wendling 2 , Aurélien Bellamy 2 , Johan Moulin Films of Ti-Zr-V were extensively studied for their use in Ultra-High Vacuum (UHV) systems like particle accelerators since the end of 20 th century [1]. The conditions of activation of a getter in a MEMS packaging and in a particle accelerator are very different [2]. This could explain why in MEMS vacuum packaging, the most used getters are Ti and Zr-Co-RE and the use of Ti-Zr-V was never reported. Thus we investigated the usefulness of the Zr-V films for MEMS packaging.

Films of Zr-V with various Zr content were co-evaporated on square silicon samples and on silicon MEMS caps. Films with high and low Zr content are nanocrystalline, while others are amorphous, as characterized by TEM. First the square samples were annealed under argon with traces of oxidizing gases at temperatures ranging from 200 °C to 400 °C and their oxygen content was measured by Nuclear Reaction Analysis (Fig. 1). Secondly the caps were sealed with a MEMS sensor at 300 °C under high vacuum and the resulting internal pressure inside the micropackages was measured by the quality factor of the MEMS and Residual Gas Analysis (Fig. 2). In both experiments, the Zr85V15 nanocrystalline film exhibited the best sorption performance compared to the Zr50V50 and Zr30V70 amorphous films.

These results show the interest of using Zr-V getter for MEMS packaging and the existence of an optimum of sorption performance between nanocrystalline and amorphous film microstructure.

Fig. 1 (

 1 Fig. 1 (left): Oxidation of annealed films; Fig. 2 (right): MEMS quality factor packaged at 300 °C