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1.
Figure 1

Figure 1. Chicken utricle hair-bundle isolation. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: Purified hair bundles, embedded in agarose, stained with phalloidin to highlight actin. Panel is 1121 x 1121 μm.
B: Purified hair bundles stained with phalloidin (red) and anti-tubulin (green). Note presence of kinocilia in most bundles but only minimal soma tubulin contamination (arrow, example of soma tubulin). Panel is 192 x 192 μm.
C: Purified hair bundles stained with phalloidin (red) and anti-tropomyosin (green). Note the scarcity of cuticular plates (arrow, example of cuticular plate). Panel is 96 x 96 μm.
D: One-dimensional SDS-PAGE separation of purified hair bundle proteins; gel was silver-stained. Migration positions corresponding to actin and major bundle bands are indicated.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
2.
Figure 2

Figure 2. Hair-bundle mass spectrometry and validation. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: Identification of creatine kinase B tryptic peptide (residues 322–342). Graphical display of monoisotopic ion peak lists generated by Mascot Distiller from tandem mass spectrometry data. Value indicated next to peak is mass-to-charge (m/z) value. Positions of y-ions (red) are indicated. Coloring coding also indicates b-ions (blue) deamidated y-ions (orange), deamidated b-ions (green), deoxidated b-ions (light blue), and unmatched ions (black). This peptide was identified as many as 7 times in a single MuDPIT experiment.
B: Distribution of bundle proteins by approximate molar amount (from intensity factor).
C-J: Validation of identifications by immunocytochemistry. For each antibody, actin counterstain is on the left, antibody labeling in the middle, and the merge (red, actin; green, antibody) is on the right. All panels are 32 x 32 μm.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
3.
Figure 4

Figure 4. Localization of creatine kinase B in hair bundles. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A-R Immunolabeling with rabbit anti-chicken B-CK antibody ().
A-C: Creatine kinase B localization in chicken utricle hair bundles. Antigen unmasking used. Projection of 7 confocal sections, each 1 μm apart.
D-E: Antibody specificity control with chick utricle. Antigen unmasking used. Excess purified B-CK protein blocks anti-B-CK labeling in stereocilia; block in kinocilium is incomplete, however.
F-I: Creatine kinase B localization in frog saccule hair cells. F, single x-y plane from a z-stack of saccular epithelium. Region used for x-z reslice is indicated by white box. G-I, reslice from z-series of F, showing cross-sections through hair cells.
J-R: Creatine kinase B localization in mouse cochlea. Antigen unmasking used. IHC, inner hair cell; OHC, outer hair cell; DC, Deiters' cell. Optical sections at three indicated depths reveal labeling in hair bundles, somas of inner hair cells, and Deiters' cells.
Scale bars: Bar in A (5 μm) also applies to B-C; bar in D (5 μm) also applies to E; bar in G (5 μm) also applies to H-I; bar in J (10 μm) also applies to K-R.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
4.
Figure 7

Figure 7. Modeling spatial buffering of ATP by creatine kinase and phosphocreatine. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: [ATP] spatial profile in absence of PCr buffering. One-dimensional source-sink model used as described in text. Parameters used: [ATP] at base fixed at 2 mM, -60 mV membrane potential, single-channel conductance of 100 pS; 3% of current carried by Ca2+, channel Po of 0.1 (dashed lines) or 0.25 (solid lines), 100% of Ca2+ pumped out by bundle PMCA (117 μM/s), glycolysis rate of 110 μM/s, diffusion coefficient for ATP in cytoplasm of 3.5 × 10−10 m2 s−1 (; ), and stereocilia radius of 200 nm. Diagram of stereocilium below indicates depletion of ATP (red); narrowing of stereocilium at base was not considered in modeling.
B: Effect on [ATP] of adding PCr buffering. Parameters: same as A with [PCr] at base fixed at 30 mM, ATP/ADP ratio of 25, K' (equilibrium constant for creatine kinase reaction) of 100, and diffusion constant for PCr of 4.9 × 10−10 m2 s−1. ATP levels remain nearly constant, even with elevated Po.
C: [PCr] gradient. JPCr/JATP ratio was 91. When Po is elevated, PCr levels fall towards stereocilium tip but remain in the millimolar range.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
5.
Figure 3

Figure 3. Detection and quantitation of hair-bundle creatine kinase B and GAPDH. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: B-CK immunoreactivity in inner ear and brain. Loading: hair bundles from 15 chicken utricles; 0.1 EE of sensory epithelium (Epithelium); 0.1 EE of utricular residue after scraping off sensory epithelium, containing stroma and nerve (Residual); ~3 μg brain protein (Brain); and 30 ng purified B-CK. Probed with anti-B-CK (top); stripped and reprobed with anti-tubulin (bottom).
B: B-CK quantitation by immunoblotting. Two samples of bundles from 5 chicken utricles each (5e) and various amounts of purified actin or B-CK were detected with actin and B-CK antibodies. Blots were scanned and analyzed to calculate B-CK and actin in the bundles from a chicken utricle. Standard curves (below) were fit with second-order (actin) or third-order (B-CK) polynomials; signals corresponding to actin and B-CK bundle samples, coded in blue or green, are indicated with arrows.
C: B-CK:actin ratio in hair bundles from gel scanning. Hair bundles were separated by SDS-PAGE; gel was stained with Coomassie blue. Left, region corresponding to B-CK and actin is shown; the band labeled as B-CK co-migrates with authentic chicken B-CK. Right, plot of Coomassie signal with distance along molecular mass axis of gel (black). Data were fit with a sum of two Gaussian curves (red); the area corresponding to B-CK was 12% of that corresponding to actin.
D: GAPDH quantitation by immunoblotting. Purified chicken GAPDH, 4% of a whole chicken utricle, or bundles from 13 chicken utricles each were detected with anti-GAPDH. The utricle and bundle lanes have approximately the same amount of total protein loaded (210 ng); the stronger intensity of the bundle band indicated that GAPDH is substantially enriched in bundles over whole epithelium.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
6.
Figure 6

Figure 6. Auditory brainstem responses and vestibular behavior in creatine kinase knockout mice. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: Typical ABRs evoked with a click stimulus (top) and with tone bursts of 8, 16 and 32 kHz (bottom) for WT, B-CK−/−, and CK=/= mice. ABRs are shown at different descending stimulus intensity levels in dB SPL. Reproducible ABRs at lower stimulation intensities in response to 32 kHz tone-bursts stimuli were not elicited from B-CK−/− and CK=/= mice; CK=/= mice also showed higher thresholds in response to 8 kHz and 16 kHz tone-burst stimuli. The vertical axis scale was adjusted for readibility at higher stimulation levels; scale bar in upper left signifies 1.5 ms and the appropriate vertical scale (indicated to left of each trace).
B: Mean auditory brainstem thresholds. CK=/= mice showed significantly elevated hearing thresholds compared to B-CK−/− and WT mice for 8 and 16 kHz (ANOVA, **p <0.01 and *p <0.05, respectively). For the 32 kHz tone burst, both B-CK−/− and CK=/= mice showed significantly elevated auditory thresholds compared to WT (ANOVA, **p < 0.01). Significance in (B) and (C) is indicated by: *, p<0.05; **, p <0.01; and ***, p<0.001.
C: Vestibular dysfunction in creatine kinase deficient mice. Applying the tail suspension and swim tests, each with scores ranging from 1 (normal vestibular function) to 5 (severe vestibular dysfunction), indicated normal vestibular function for WT mice (n=30). B-CK−/− single knockout mice (n=17) showed a mild vestibular dysfunction; dysfunction in CK=/= double knockout mice (n=17) was more severe.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.
7.
Figure 5

Figure 5. Creatine kinase inhibition increases ATP hydrolysis and slows adaptation in hair bundles. From: Hair Bundles Are Specialized for ATP Delivery via Creatine Kinase.

A: Creatine kinase and PMCA are major sources of ATP synthesis and hydrolysis, respectively, in hair bundles. ATP consumption can be monitored indirectly with Mg Green; because the affinity of Mg2+ for ADP is much less than for ATP, hydrolysis of ATP will increase free Mg2+ levels. Rephosphorylation of ADP by creatine kinase will keep free Mg2+ levels low; inhibition of creatine kinase (by DFNB) should elevate Mg2+, increasing Mg Green fluorescence.
B: ATP levels in isolated bullfrog hair cells after treatment with DNFB or carboxyeosin. ATP in ~7 cells was measured with luciferin/luciferase assay. These experiments are representative of three independent experiments.
C: Average rate of increase (initial slope) of Mg Green fluorescence from first six points (12 min) of each cell, individually fit with single-exponential functions (n=19). The difference between bundles and somas is significant (**, p<0.02).
D: Transduction currents before (left) and after (middle) 30 min treatment with 100 μM DNFB; right traces show transduction currents after 500 μM Ap5A was added to the apical solution as well
E: Transduction currents before (left) and after (right) 30 min treatment with 5 mM 2DOG; 5 mM pyruvate was included in apical and basal solutions.
F: Transduction currents before (left) and after (right) 30 min treatment with 100 μM DNFB and 5 mM 2DOG; 5 mM pyruvate was included in apical and basal solutions.
G: Fast adaptation rate constants before (control; n=32) and 30 min post-perfusion with control salines (n=10); 100 μM apical and basal DNFB (n=10); 100 μM DNFB apical and basal followed by 500 μM apical Ap5A (n=3); 5 mM 2-deoxygluycose and 5 mM pyruvate in glucose-free salines (n=5); 100 μM DNFB, 5 mM 2-deoxygluycose, and 5 mM pyruvate in glucose-free salines (n=7). Significance (two-tailed Student's t-test) indicated by: *, p<0.05; **, p<0.02. Significance for the Control-DNFB comparison was p=0.056.
H: Slow adaptation rate constants; same epithelia as (G). Significance for the Control-DNFB comparison was p=0.091.

Jung-Bum Shin, et al. Neuron. ;53(3):371-386.

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