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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3–10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

Streszczenie

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1–2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Wprowadzenie

The hair cells of the inner ear are the sensory cells that detect sound and covert the mechanically stimuli in electrical signals, which are ultimately interpreted by our brain. These cells have a staircase-shaped bundle of three rows of actin-based filaments, known as stereocilia, which protrude from their apical region1,2. The mechanical stimuli deflect the stereocilia filaments toward the longest row and trigger the opening of the mechanotransduction (MET) channels3. The opening of the MET channels leads to an influx of cations that depolarizes the cell and consequently signals the release of synapse vesicles at the basal region of the hair cell.

The biophysical properties of the MET channel essential for hearing have been extensively characterized. Among other properties, these channels are cationic selective and have a relatively large conductance (150–300 pS in low Ca2+)4,5,6,7,8,9,10. Remarkably, large fluorescent molecules such as FM1-43 and Texas Red-labeled aminoglycosides are permeant blockers of the MET channel, resulting in their accumulation in the hair cell body that can be visualized using fluorescence microscopy11,12,13,14. Conversely, the molecular identity and the structure of the MET channel and its permeation pathway have remained elusive. Increasing experimental evidence indicates that the transmembrane-like channel protein 1 (TMC1) is a component of the MET channel in mature hair cells15,16,17,18,19. Mutations in the transmembrane-like channel 1 (TMC1) alter the MET channel properties19,20,21,22 and cause deafness. In addition, TMC1 localizes to the site of the MET channel18,23 and interacts with the tip-link responsible for transmitting the mechanical force to the MET channel24,25. Furthermore, recent bioinformatics analysis has identified the TMC proteins as evolutionary related to the mechanosensitive channels TMEM63/OSCA proteins and the TMEM16 proteins, a family of calcium-activated chloride channels and lipid scramblases26,27,28. A structural model of TMC1 based on the relationship between these proteins revealed the presence of a large cavity at the protein-lipid interface27. This cavity harbors the two TMC1 mutations that cause autosomal dominant hearing loss (DFNA36)27,29,30,31,32, and selective modification of cysteine mutants for residues in the cavity alter MET channel properties28, indicating that it could function as the permeation pathway of the MET channel. The large size of this predicted cavity in TMC proteins could explain the ability of large molecules to permeate the MET channel. To test the prediction that the MET channel contains an unusually large permeation pathway and to push the limits of the size of the cavity observed in TMC1, we developed a protocol to perform uptake experiments in the organ of Corti explants with a larger molecule, 3 kDa dextran fluorescently labeled with Texas Red.

Dextran is a complex branched polysaccharide composed of many D-glucose molecules bound by alpha-1,6 glycosidic linkages. Its high solubility in water, low cell toxicity, and bioinertity make it a versatile tool to study several cellular processes. In addition, dextran is available in a wide range of sizes and fluorescently labeled with fluorophores of several colors. Fluorescently labeled dextrans are commonly used in cell and tissue permeability research33,34, to study endocytosis in multiple cellular systems35,36, and for neural tracing37,38. In the auditory field, dextran molecules have also been used to assess the disruption of the cell-cell junction and loss of the auditory sensory epithelium integrity after exposure to intense noise in the chinchilla organ of Corti39,40.

In this work, we exploited the properties of some of the smallest (3 and 10 kDa) fluorescent dextrans to perform uptake experiments in murine inner ear hair cells and explore the size of the permeation pathway of the inner ear hair cell MET channel. In addition, we used a laser-scanning confocal microscope (LSM) 880 equipped with an Airyscan detector to visualize and localize fluorescent dextran at the stereocilia and the cell body of auditory hair cells.

Protokół

The animal care and experimental procedures were performed following the guidelines for the Care and Use of Laboratory Animals, which were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke (Animal protocol #1336 to KJS).

1. Mice

  1. Set a couple of breeding pairs of C57BL/6J wild-type to breed in the animal facility to control the date of birth of the litters and keep track of the age of the pups.

2. Cochleae Dissection

  1. Set a clean space close to a stereomicroscope to perform the dissections (Figure 1A). Use 70% ethanol to clean the space and surroundings and place a clean bench pad. A Medical Pathological Waste (MPW) plastic bag would be required to discard the animal carcasses.
  2. Prepare several 35 mm dishes with some Leibovitz's L15 media.
    NOTE: Leibovitz's L-15 cell media contains 1–2 mM Ca2+, which is required to maintain the integrity of the tip-links, and contains essential amino acids, vitamins, and sodium pyruvate to improve cell health and survival. Serum was excluded to avoid experimental variability due to its poorly defined composition and potential interference with the dextran.
  3. Euthanize postnatal-day-6 (P6) mice by decapitation.
    NOTE: 6 day old mice are somewhat resistant to inhalant anesthetics. Although isoflurane or prolonged CO2 exposures (up to 50 min) may be used for euthanasia, a secondary physical method is recommended to ensure death.
  4. Use surgical scissors to remove the skin of the skull by making a superficial cut from the anterior to the posterior end and across the external auditory canals.
  5. Fold the skin towards the nose to expose the cranium (Figure 2B).
  6. Make an incision from the back to the front of the skull and across the eye line (Figure 2B-C).
  7. Separate the skull in two halves and remove the brain with the use of a small spatula to expose the temporal bones (Figure 2C-D).
  8. With small scissors, cut around the temporal bones, and excise the tissue.
  9. Place both temporal bones in a 35 mm dish and make sure they are covered with L15 media (Figure 2E).
    NOTE: The following steps are performed under the stereomicroscope. A black background usually helps to visualize the tissue during the fine dissection steps.
  10. Under a stereomicroscope equipped with a widefield eyepiece (a 10X magnification power (WF10X) and an external alternating current (AC) halogen light source), remove the surrounding cochleae tissue, semicircular canals, and vestibular organs with surgical forceps (Figure 2F).
  11. To allow the dextran and L15 media to enter the cochlear duct, perform two puncture bounds on the dissected cochleae, one on the round window and other at the apical cochlear region.
  12. Add 300 mL of Leibovitz's L15 media in each well from a 9-well glass depression plate.
  13. Place at least three dissected cochleae on each well.

3. Dextran Labeling

  1. Reconstitute the dextran in Hanks' balanced salt solution without Ca2+ and Mg2+ (HBSS-CFM) at a final concentration of 10 mg/mL. This stock solution must be aliquoted in opaque black tubes (protected from light) and stored at -30 °C until use.
    NOTE: The use of lysine-fixable dextran is critical for a successful outcome of this protocol.
  2. Prepare each dextran at a final concentration of 2 mg/mL in 500 mL of Leibovitz's L15 media.
  3. Remove the media from the cochlea and add Leibovitz's L15 media containing the dextran of interest at a final concentration of 2 mg/mL.
    NOTE: Although a proportion of the MET channels are open at rest41,42, the dextran incubation was performed with a gentle shaking of the explants to increase the open probability of the MET channel.
  4. Incubate at room temperature for 2 h with gentle shaking (25 rpm) by using a 3-dimensional shaker with a tilted angle of 25°.
    NOTE: Fluorescently labeled dextran must be protected from light when possible. To protect the dextran during the 2 h incubation, place the 9-well glass plate inside a cell culture P150 dish wrapped in aluminum foil.

4. Sample Preparation for Imaging

  1. After incubation with the dextran, wash the tissue for 2 min twice with media and once with HBSS.
  2. Incubate the tissue at room temperature for 30 min with 4% paraformaldehyde in Hanks' balanced salt solution (HBSS).
    CAUTION: Exposure to formaldehyde can be irritating to the eyes, nose, and upper respiratory tract. In certain individuals, repeated skin exposure to formaldehyde can cause sensitization that may result in allergic dermatitis. Formaldehyde is a known human carcinogen and a suspected reproductive hazard.
  3. Quickly and gently wash the fixed tissue twice with HBSS to remove the paraformaldehyde.
    NOTE: Decalcification of the temporal bones is not needed at this developmental stage of the cochlea.
  4. Remove the spiral ligament and the tectorial membrane with fine tip forceps to dissect the organ of Corti (Figure 2G).
  5. Remove all the small pieces of tissue and wash the tissue with HBSS.
  6. Permeabilize the tissue in 0.5% Triton X-100 in PBS containing fluorescently-labeled phalloidin (conjugated to green or red when testing the uptake of TR- or FITC-labeled dextran, respectively) at a 1:200 dilution for 30 min to label F-actin and visualize the actin-based stereocilia.
  7. Wash the tissue 2–3 times for 2 min each time with HBSS buffer to remove the excess of triton and phalloidin, and once with PBS to remove the salts.
  8. Mount the organ of Corti tissues on a microscope slide and cover it with mounting media.
    NOTE: When mounting the tissue, make sure that the side of the tissue containing the hair cell stereocilia is facing the coverslip.
  9. Remove any potential bubbles generated during the addition of the mounting media and prevent the generation of new bubbles during the placement of the coverslip.
    NOTE: Aspirate with a pipette any bubble generated during the addition of the mounting media. To prevent air bubbles from being trapped under the coverslip, place an edge of the coverslip close to the sample and carefully and slowly lower the coverslip over the tissue using forceps or a pipette tip.
  10. Cover the tissue in mounting media with a glass coverslip (Figure 2H).
    NOTE: Objectives with a numerical aperture above 0.4 are designed to use #1.5 coverslips (0.17 mm thickness). Using the wrong coverslip may have severe implications for the intensity and quality of the images.
  11. Incubate the mounted tissue overnight at room temperature to let the mounting media dry and store the slides at 4 °C until imaging.

5. Image Acquisition and Image Processing

NOTE: The confocal images were taken with a LSM 880 confocal microscope equipped with a 32 channel Airyscan detector in the super-resolution (SR) mode43 and a 63x objective.

  1. Add a small drop of immersion oil on the objective.
  2. Place the microscope slide containing the mounted tissue sample in the microscope stage with the glass coverslip facing the immersion oil.
  3. Focus on the sample and set the imaging parameters using the image acquisition software.
  4. Use identical image acquisition settings and optimal parameters for x, y, and z resolution for each independent experiment. A piezo-driven focus system is required to quickly and precisely move the objective when acquiring the z-stack of images.
    NOTE: To image the entire apical region of the hair cells, collect a z-stack of images from the stereocilia to the apical half of the hair cell body using the optimal settings. It is crucial to collect a large z-stack along the hair cell to assure the imaging of the vesicle-like particles.
  5. Use the image acquisition software to process the raw confocal images using the Airyscan 3D reconstruction algorithm with the automatic default deconvolution filter settings.
  6. Open the confocal images in an image processing software to adjust the brightness and contrast, add the scale bar, and export the images for the final figures.

Wyniki

We observed robust and specific labeling of hair cells after 2h incubation of organ of Corti explants from wild-type postnatal-day-6 (P6) mice with 3 kDa dextran fluorescently labeled with Texas Red (dextran-TR) (Figure 2A-B). Dextran labeling was observed in both inner and outer hair cells (IHC and OHC) at the basal, middle, and apical regions of the organ of Corti (Figure 2B).

Fluorescently labeled ...

Dyskusje

This protocol describes how to perform uptake experiments in murine organ of Corti explants with 3 kDa dextran Texas Red. The goal of this method is to test whether molecules larger than others previously tested were also able to specifically label auditory hair cells and permeate through the MET channel. Similar experimental protocols have been previously used to evaluate the permeability of hair cells to other fluorescent dyes such as FM1-43 (0.56 kDa)12,19

Ujawnienia

The authors have nothing to disclose.

Podziękowania

We thank Vincent Schram from the NICHD microscopy and imaging core for assisting in the confocal image acquisition, and Tsg-Hui Chang for invaluable help with colony management and mice care. This research was supported by the Intramural Research Program of the NINDS, NIH, Bethesda, MD, to K.J.S. A.B. was supported by the Intramural Research Program of the NINDS, NIH, and by a Robert Wenthold Postdoctoral Fellowship from the intramural research program of the NIDCD.

Materiały

NameCompanyCatalog NumberComments
#1.5 glass coverslips 18mmWarner Instruments64-0714
Alexa Fluor 488 PhalloidinThermoFisherA12379
Alexa Fluor 594 PhalloidinThermoFisherA12381
alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objectiveCarl Zeiss420780-9970-000
Amiloride hydrochlorideEMD MILLIPORE129876
Benchwaver 3-dimensional RockerBenchmarks scientificB3D5000
C57BL/6J wild-type micestrain 000664The Jackson Laboratory
Cell impermeant BAPTA tetrapotassium saltThermoFisherB1204
Dextran, Fluorescein, 10,000 MW, Anionic, Lysine FixableThermoFisherD1820
Dextran, Texas Red, 10,000 MW, Lysine FixableThermoFisherD1863
Dextran, Texas Red, 3000 MW, Lysine FixableThermoFisherD3328
Formaldehyde Aqueous Solution EM GradeElectron microscopy science15710
HBSS, calcium, magnesium, no phenol redThermoFisher14025
HBSS, no calcium, no magnesium, no phenol redThermoFisher14170
Image J or FIJINIHhttp://fiji.sc/
Immersol 518F oil immersion mediaCarl Zeiss444970-9000-000
Leibovitz's L-15 Medium, GlutaMAX SupplementThermoFisher31415029
neomycin trisulfate salt hydrateSigmaN6386
PBS (10X), pH 7.4ThermoFisher70011069
Phalloidin-CF405MBiotium00034
ProLong Diamond antifade mounting mediaThermoFisherP36970
superfrost plus microscope slideFisherbrand22-037-246
Triton X-100SigmaT8787
Zen Black 2.3 SP1 softwareCarl Zeisshttps://www.zeiss.com/microscopy/us/products/microscope-software/zen.html

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