Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear.

Plasmids and biolistic transfection

For live-cell imaging experiments, human ACTB cDNA ( NM_001101.3 , identical amino acid sequence to mouse β-actin) cloned into the Green FP expression vector (BD Biosciences, San Jose, CA) was used to prepare gold particles for biolistic transfection as previously described 42 with minor modifications. Specifically, we used antibiotics in the culture media (see Organ culture and live imaging below) and reduced the BioRad Helios Gene Gun firing pressure to 100 p.s.i. in order to reduce damage caused by biolistic transfection. The p.G13R and p.G63D substitutions of β-actin were introduced into pEGFP-β-actin using site-directed mutagenesis (Stratagene, La Jolla, CA) with primers 5′-CGTCGTCGACAAC C GCTCCGGCATGTG-3′, 5′-CACATGCCGGAGC G GTTGTCGACGACG-3′, 5′-CCCAGAGCAAGAGAG A CATCCTCACCCTGAA-3′ and 5′-TTCAGGGTGAGGATG T CTCTCTTGCTCTGGG-3′, respectively (point mutation in bold). pDendra2-β-actin was cloned using InFusion (Clontech, Mountain View, CA) with primers 5′-GTGTACAAGACTCGAGCCACCATGGATGATGATATCG-3′ and 5′-TAGATCCGGTGGATCCCTAGAAGCATTTGCGGT-3′ to amplify the cDNA of ACTB and insert it between the XhoI and BamHI sites of pDendra2-C (Clontech, Mountain View, CA). The DNA sequences of all plasmid inserts were verified by Sanger sequencing.

Organ culture and live imaging

C57Bl6/J pups were killed by decapitation at postnatal day 2–5 in accordance with National Institutes of Health Institutional Animal Care and Use Committee-approved guidelines under animal study protocol #1263. Utricles were dissected in L-15 media (Life Technologies, Carlsbad, CA, USA) and cultured on 2 mg ml −1 collagen matrices in DMEM/F-12 with 20 mM L -glutamine, 7% fetal bovine serum, and 1 unit per ml penicillin-G. One hour before biolistic transfection, growth media was replaced with antibiotic-free media. Transfections were done using a Helios gene gun and endotoxin-free plasmid DNA (NucleoBond Xtra EF, Macharey-Nagel). Twenty-four hours post transfection, cultures were transferred to phenol-red free media supplemented with penicillin-G and mounted under a platinum and nylon harp 46 at 37 °C in a humidified 6% CO 2 atmosphere with the stereocilia facing the coverglass and objective. Cells transfected with pEGFP-β-actin were live-imaged on a Zeiss Cell Observer spinning disc microscope (× 63 plan-apochromat, NA 1.4) for 62–92 h. Z-stack volumes (138 × 0.4 μm) were captured at 1-h intervals. For photoconversion experiments with pDendra2-β-actin, transfected cultured cells were imaged for 24 h and z-stack volumes were captured at 30 min intervals. Dendra2-β-actin was converted by exposing a defined region of interest to 405 nm laser light for 15 × 1-ms bursts at 6% of the total laser power (UltraView, Perkin Elmer, Akron, OH, Laser Module 2). Post-acquisition image analyses were performed using ImageJ ( http://imagej.nih.gov/ij/ ) and Volocity (Perkin Elmer, Akron, OH, USA). For automated analysis of dendra2-β-actin experiments ( Fig. 5d ), the red photoconverted populations of dendra2-β-actin and the green non-photoconverted dendra2-β-actin cuticular plate were identified mathematically. The distance from the centroid of the red population to the edge of the cuticular plate was measured for each time point.

Fixed-cell imaging

Mutant expression constructs pEGFP-β-actin G63D and pEGFP-β-actin G13R , the wild-type EGFP-β-actin control and the EGFP-only control were biolistically transfected into cultured utricles from neonatal mice (P2–P5) and fixed in 4% paraformaldehyde at 4 and 24 h post transfection for imaging 17 18 33 . After fixation, samples were washed in phosphate-buffered saline, counterstained with rhodamine-conjugated phalloidin and mounted in ProLong Antifade Gold (Life Technologies).

Multi-isotope imaging mass spectrometry

Mouse pups born to normal mothers were switched at birth to nurse from mothers who had been fed for a month or more with food containing 15 N-leucine (∼1.25% relative to 14 N-leucine). Pups were killed after 4 or 15 days. Utricles were fixed overnight at 4 °C in 4% formaldehyde+10% glutaraldehyde in cacodylate buffer, then processed for plastic embedding and thin sectioning. We performed MIMS as described previously 47 48 . Briefly, an accelerated beam of Cs + ions, focused to a 30-nm spot, was scanned across the surface of a thin section, sputtering molecules from the surface. Anionic molecules were accelerated back and analysed with a double sector mass spectrometer. 15 N was detected as the cyanide ion 12 C 15 N − at mass 27 and compared with 12 C 14 N − at mass 26 for each pixel in the scanned image. Incorporation was calculated from the natural abundance of 15 N and the relative abundance of 15 N in the food 26 . To show incorporation as a percentage in the image, we used a hue saturation intensity transformation in which the hue represents percentage and the intensity indicates reliability.

Supplementary Material Supplementary Information Supplementary Figure 1 Supplementary Movie 1 Montage of all cells classified as “steady-state tip localization”. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped to identical size without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 2 Montage of cells classified as “stereociliogenesis” – group 1. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped to identical size without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 3 Montage of cells classified as “stereociliogenesis” – group 2. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped to identical size without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 4 Montage of cells classified as “damaged” – group 1. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 5 Montage of all cells classified as “damaged” – group 2. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 6 Montage of all cells classified as “damaged” – group 3. Displayed as a single movie, each region of interest is presented in a single box as a maximum intensity projection. All movies were cropped without changing magnification and the brightness and contrast was uniformly normalized to 0.3% saturation for each frame of each movie using the “Enhance contrast – normalize” function in ImageJ. Supplementary Movie 7 Movie of cell shown in Figure 5B