Detection of TMCC2 using different antibodies in transfected cells

In order to identify tools suitable for the characterization of the Tmcc2^-/- knockout mice, I tested two different antibodies against TMCC2 in respect to their reactivity and potential crossreactivity with other TMCC family members in transfected HEK293T cells.

Both the ARP42418_P050 and the 25042-1-AP antibodies recognized the N-terminal segment of TMCC2 protein but their target antigens are located between amino acids 244-295 and 1-233 respectively. Here, I have transfected HEK293T cell line with TMCC1,

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TMCC2 or TMCC3 and later stained with the ARP42418_P050 and the 25042-1-AP antibodies (Fig. 3). The 25042-1-AP antibody strongly and specifically labeled TMCC2 transfected cells (Fig. 3H). No staining was observed in untransfected cells or cells transfected with TMCC3 while only weak non-specific binding was seen in cells transfected with TMCC1. The cells transfected with TMCC2 also stained with ARP42418_P050 (Fig.

3G) but the signal was not nearly as bright as that of the 25042-1-AP.

Figure 3. Anti-TMCC2 antibodies specifically recognize TMCC2 in transfected HEK293T cells. (A-C) Untransfected HEK293T cells. In the remaining panels cells were transfected with: (D-F) TMCC1, (G-I) TMCC2, (J-L), or (J-L) TMCC3. The cells are stained with (A, D, G, J) anti-TMCC2 (ARP42418_P050), (B, E, H, K) anti-TMCC2 (25042-1-AP) or (C, F, I, L) anti-FLAG. Note the strong TMCC2 specific binding of 25042-1-AP. In all panels the nuclei were stained with DAPI. Scale bar: 50 µm, 20x objective, scale bar for zoomed image: 10 µm, 20x objective.

In last panel, I stained cells with anti-FLAG (ROCK600-401-383) which stained cells transfected with TMCC2 (Fig. 3I) and some unspecific staining was shown in cells transfected with TMCC1 (Fig. 3F). However, no signal was observed in untransfected cells, cells transfected with TMCC1 and TMCC3 stained by the ARP42418_P050 antibody. Based

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on the experiment, both antibodies tested against TMCC2 looked promising. However, none of the available antibodies are suitable for the detection of the erythroblast specific isoform as this variant does not have the N-terminal portion of the full length TMCC2 which contains the antigenic sequences used to produce these antibodies.

4.1.3 Confirmation of successful disruption of the Tmcc2 gene

In order to confirm the deletion of Tmcc2 in the knockout mice, a genomic DNA fragment was amplified by PCR, with the use of primers flanking the designed target site and sequenced using Sanger sequencing (Fig. 4A). In order to eliminate the need for sequencing during standard genotyping, I worked out the optimal conditions to detect an 11 bp difference between the alleles by PCR and agarose gel electrophoresis (Figure 2B). TMCC2 is expressed in the nervous system (Hopkins, Sáinz-Fuertes, and Lovestone 2011). In order to check whether the deletion introduced in the Tmcc2 knockouts was sufficient to abolish the expression of the TMCC2 protein, I prepared protein extracts from the brains of Tmcc2^+/- and Tmcc2^-/- mice at P4 and performed a western blot analysis with a TMCC2-specific antibody.

A band corresponding to a protein of expected size of ~80 kDa was present in controls but absent in the knockout mice (Fig. 4B). As an additional antibody specificity control, I transfected HEK293T cells with a Tmcc2 cDNA construct with a hemagglutinin (HA) tag and confirmed that a specific band of ~80 kDa was present in the extracts from transfected cells (Fig. 4B).

In order to check for potential embryonic lethality of the Tmcc2^-/- mice, I calculated the ratios of mice for different genotypes and found Mendelian pattern of inheritance with expected genotype ratios for a cross of two heterozygous parents. For initial phenotypic characterization of the mice, I looked closely for any visible differences among the wild-types and the knockouts. The adult mice were responding to unexpected sounds with a normal flinching reaction, had no apparent problem maintaining balance while walking, climbing the rods of a cage lid or when placed on a thin wooden beam. None of the knockout mice displayed the circling behavior that is characteristic for mutations that affect the vestibular hair cells.

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Figure 4. Expression of TMCC2 abolished in Tmcc2^-/- mice. (A) Validation of the deletion by Sanger sequencing of a PCR product obtained from heterozygous founder genomic DNA with primers flanking the target sequence (the arrow points to the position where the sequences of the two alleles diverge). (B) Genotyping PCR result with different genotypes.

The knockout band is 11 bp smaller than the wild-type one. The absence of a specific ~80 kDa TMCC2 protein in the Tmcc2^-/- whole brain extracts. The presence of a specific ~80 kDa TMCC2 protein band of expected size in the transfected HEK293 cells.

4.1.4 Expression of TMCC2 in hair cells of mice

To determine the expression pattern of TMCC2 in hair cells, I have stained organ of Corti preparations of Tmcc2^+/+ and Tmcc2^-/- mice with the rabbit antibody directed against TMCC2 (ARP42418_P050) and with phalloidin to stain the actin of the stereocilia.

According to the previous preliminary observations with an inferior antibody HPA01425 that requires antigen retrieval, the TMCC2 protein localizes in the cell body rather than stereocilia. In whole-mount organ of Corti preparations from wild-type mice TMCC2 signal was localized to the cell body also including the apical surface of the cell and the level below the actin-rich cuticular plate in which the rootlets of stereocilia are anchored. No signal was detected in the hair bundles. Importantly, the staining of TMCC2 was completely absent in the knockout mice (Fig. 5D and 5F). The morphology of the hair bundles was normal in wild-type and knockout mice (Fig. 5A and 5B).

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Figure 5. Expression of TMCC2 is abolished in hair cells of Tmcc2^-/- mice. (A, B) Inner and outer hair cells were visualized with phalloidin (blue) in confocal analysis at the hair bundle level (A) of Tmcc2^+/+ mice and (B) of Tmcc2^-/- mice. (C, D) Same inner and outer hair cells stained with anti-TMCC2 antibody (red) visualized at cuticular plate level. (C) dot like staining in Tmcc2^+/+ outer hair cells, (D) Tmcc2^-/- mice show no staining. (E, F) At the cell body level the TMCC2 signal fills both inner and outer hair cell in (E) Tmcc2^+/+ mice whereas (F) no staining is visible in Tmcc2^-/- mice. (NWT=12, NKO=9) Scale bar: 10 µm.

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4.1.5 Morphology of the hair bundles and the presence of CDH23 in stereocilia To investigate potential impact of the loss of TMCC2 on the morphology of the hair bundle, I stained hair cells with antibodies against tubulin and CDH23. Tubulin stains the kinocilium which is not crucial for mechanotransduction but functions in the development of the stereocilia and helps in the staircase arrangement of the hair bundles (Fig. 6C and 6D).

Figure 6. Tmcc2 knockout HC at P5 display normal morphology. (A, B) Inner and outer hair cells were visualized with anti-Cadherin23 (red) antibody and anti-Phalloidin (blue) in confocal analysis at stereocilia level in (A) Tmcc2^+/- and (B) Tmcc2^-/- mouse. and shows normal distribution of Cadherin 23. (C, D) Tubulin (red) and anti-Phalloidin (blue) at stereocilia level (C) in Tmcc2^+/- mouse and Tubulin stains the kinocilium of the hair cell which helps in the growth of the stereocilia in a staircase pattern, (D) in Tmcc2^-/- mouse shows normal distribution of Tubulin at P5. Scale bars: 10 µm.

The kinocilium disappears in the mature hair cells. CDH23 plays a developmental role as a component of transient lateral links between stereocilia and between stereocilia and the kinociliun during the development of the bundle. It is also a component of the upper end of the tip link and one of the proteins critically required for mechanotransduction. The morphology of hair cells at P5 in the Tmcc2 knockout mice, the positioning of kinocilia relative to stereocilia and the localization of CDH23 are normal (Fig. 6).

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4.1.6 Localization of apical markers – EPS8 and EPS8L2

To investigate the morphology of hair bundle, whole mount sections of Tmcc2^+/+ and Tmcc2^-/- were prepared and stained with phalloidin which stains actin in the hair bundle. The hair cells were also stained with antibody directed against EPS8 and EPS8L2, two proteins responsible for the regulation of hair-bundle structure and maintenance. EPS8 controls the length of the stereocilia and plays a role in the bundling and capping of actin filaments localized in longest row of stereocilia (Fig. 7B, 7E and 7H). EPS8L2 has a complementary role in the maintenance of hair bundles and EPS8L2 is localized at the tips of short and intermediate rows of stereocilia (Fig 7C, 7F and 7I). The IHCs in knockout mice exhibit normal morphology of the hair bundles and typical distribution of EPS8 and EPS8L2 staining, lending further support to the notion that the normal asymmetric polarization of the hair bundle is maintained in the Tmcc2^-/- hair cells.

Figure 7. Tmcc2 knockout IHC bundles at P3 display normal morphology. (A-C) EPS8 (green) and EPS8L2 (red) show asymmetric distribution between stereocilia of the longer and shorter rows in knockout IHC bundles at P3. (D-F) CDH23 (green), a component of lateral links and tip links, is present throughout the bundle at this developmental stage, including the tips of stereocilia labeled with EPS8L2 (red). (G-I) EPS8 and EPS8L2 in wild type IHC bundles at P3. Scale bar (A-C): 2 µm, (D-I): 5 µm.

4.1.7 Localization of TMCC2 in hair cells

To study the localization of the TMCC2 protein, I stained the hair cells with antibody directed against MYO7A, a cell body marker of hair cells which is a motor protein involved

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in the regulation the growth of stereocilia. The staining of MYO7A is described to localize in the cell body and plasma membrane surrounding the OHCs and IHCs (Fig. 8C and 8F).

TMCC2 as described previously localized to the ER (Zhang et al. 2014) and in the cell body of the hair cells. The co-staining of TMCC2 and MYO7A revealed that TMCC2 is not only expressed in ER but also in the cell membrane. The expression of TMCC2 was in the cell membrane of both OHCs and IHCs (Fig. 8B and 8E).

Figure 8. Localization of TMCC2 in the cell membrane and not just ER. (A-C) Inner hair cell was visualized under confocal microscope with antibody against MYO7A (green) and TMCC2 (red) at P3 (B) TMCC2 protein stains the plasma membrane and the cell membrane of the inner hair cell as (C) MYO7A stains the plasma membrane surrounding the inner hair cell. (D-F) Outer hair cell was visualized with antibody againstMYO7A (green) and TMCC2 (red) in confocal analysis at P3 (E) TMCC2 protein stains both the plasma membrane and cell membrane of the outer hair cell as (F) MYO7A stains the plasma membrane surrounding the outer hair cell. Scale bar: 0.2 µm.

4.1.8 Analysis of supernumerary OHCs in Tmcc2^-/- mice

While analyzing cochlear histology, I noticed unusually long stretches of extra row of OHCs and occasional extra IHCs. First, I observed supernumerary OHCs at P18 stained with antibodies against MYO7A. The incidence of extra row of OHCs appeared to be higher in the knockout mice compared to the age-matched wild type controls of C57BL6/J background.

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While the presence of some supernumerary hair cells is considered normal in mice, such phenotype could potentially be a consequence of the loss of TMCC2. For example, disruption of Notch signaling may cause the duplication of both inner hair cells and inner phalangeal cells, mainly due to mutations in the two Notch modifiers Lfng and Mfng (Basch et al. 2016).

Figure 9. Supernumerary OHCs observed in Tmcc2^-/- mice. (A-F) Inner and outer hair cell stained with anti-MYO7A to visualize the extra rows of hair cells at P18 in (A-C) Tmcc2^+/+

and (D-F) Tmcc2^-/- mice. The yellow arrows indicate the extra rows of outer hair cells. Scale bar: 20 µm.

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Figure 10. Increased number of supernumerary OHCs is not associated with the Tmcc2

-/- genotype mice. (A-F) Inner and outer hair cells stained with anti-MYO7A for the visualization of supernumerary outer hair cells at P4 in (A-C) in Tmcc2^+/+ and (D-F) Tmcc2 -/-mice. The yellow arrow indicates the extra rows of outer hair cells and occasional inner hair cells. Scale bar: 10 µm. Three mice each were used for counting hair cells of wildtype and knockout.

Moreover, inbred strains of mice differ slightly in the average number of supernumerary hair cells. Since the Tmcc2^-/- line was raised on a mixed background and crossbred to C57BL6/J for three generations it was important to determine whether the percentage of supernumerary OHCs was associated with the lack of TMCC2 or with the genetic background of the mice. I quantified this in organs of Corti from wild-type and knockout mice of the same mixed background, stained with Phalloidin and Myo7a at two different ages: P4 and P18 (Fig. 9-11) I counted nearly 26,000 OHCs and calculated the percentage of extra OHCs to OHCs and found that the occurrence of extra row of OHCs is not associated with the genotype of the mice but is typical for the genetic background of our colony.

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Figure 11. The percentage of supernumerary OHCs is variable and is not associated with the Tmcc2^-/- genotype. The fraction of extra outer hair cells (eOHCs) to OHCs were counted manually at P4 and P18 in Tmcc2^+/+ (N=3) and Tmcc2^-/- (N=3) mice. The total number of cells counted (eOHCs/OHCs) were: P4 WT=2434/7526, P4 KO=2712/8324, P18 WT=1048/3413 and P18 KO=2156/7300.

4.1.9 Expression of TMCC3, a close paralog of TMCC2

The results presented so far indicate, that despite strong expression of TMCC2 in hair cells this protein is not required for their development or basic function. I found this surprising and looked for a possible molecular explanation in a potential compensation by other family members. According to the Shared Harvard Inner Ear Database (SHIELD) TMCC1 mRNA is absent in hair cells but low levels of TMCC3 mRNA were detected. In order to confirm the expression of TMCC3 in Tmcc2^+/+ and Tmcc2^-/- mice, I have stained cells with antibodies recognizing TMCC2 (ARP42418_P050) and TMCC3. I observed that TMCC3 is expressed in both IHCs and OHCs of inner ear but not in the same plane as TMCC2. The staining of TMCC3 was in the pericuticular level. While the localization of TMCC3 in the Tmcc2 knockout hair cells is similar to wild-type controls the intensity of the staining is much higher (Fig. 12K). The significance of this discovery is further explained in the Discussion.

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Figure 12. TMCC3, a close paralog of TMCC2 is expressed in the hair cells. (A-C) Inner and outer hair cells visualized under confocal microscope stained with anti-TMCC2 (red) and anti-TMCC3 (blue) at cell body level. TMCC3 does not localize with TMCC2 instead localizes below the cuticular plate as shown in (D-F) in Tmcc2^+/+ mouse. (G-L) Inner and outer hair cells stained with anti-TMCC2 (red) and anti-TMCC3 (blue) in Tmcc2^-/- mouse shows normal distribution of TMCC3 in the absence of TMCC2 protein. Scale bar: 10 µm

4.1.10 Summary of the findings regarding TMCC2 in hair cells 1. The expression of TMCC2 was abolished in knockout mice.

2. Inner and outer hair cells display normal morphology verified using different markers:

MYO7A, CDH23, Tubulin, EPS8 and EPS8L2.

3. The localization of TMCC2 is not just in the endoplasmic reticulum (ER) but also in the cell membrane.

4. Supernumerary outer hair cells are present in mice from our colony but are not associated with the Tmcc2^-/- genotype.