Stereocilia are one particular example in which elaborate protein ensembles are maintained at distances that can be up to 100 m from their sites of delivery and uptake
Stereocilia are one particular example in which elaborate protein ensembles are maintained at distances that can be up to 100 m from their sites of delivery and uptake. exhibit high mobility with a diffusion coefficient of 0.1C0.2 m2/s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal. and planes and imaged within 15 min of being removed Procyanidin B2 from the 37C chamber. A beam spot of 1 1.2 m diameter produced with a 488 nm laser was positioned over the hair bundle before bleaching a portion of the Rabbit Polyclonal to Collagen III stereocilia in the bundle. A preexposure frame was collected, and sequential frames after the bleaching pulse were collected during the recovery. A region of interest within the stereocilia bleach area was selected, and the average fluorescence intensity was measured for each time point. Background fluorescence was measured and subtracted from these values, and they were corrected for the fluorescence fading during image acquisition using, as reference, the fluorescence intensity in a neighboring nonphotobleached region of the hair cell. The recovery after bleaching was fit using Origin: (1 Procyanidin B2 ? is the fluorescence intensity, is the end value of the recovered intensity, is the fitted parameter, is the time after the bleaching pulse, and is the initial fluorescence intensity. Simulation of diffusion of PMCA2 in the stereocilia membrane. The program is available from http://sahara.lbl.gov/sandrews/software.html with a description of the algorithm (see also http://www.pdn.cam.ac.uk/groups/comp-cell/has been given previously (Andrews and Bray, 2004). Simulations were run on an Apple Computers (Cupertino, CA) Power Mac G5 (dual, 2 GHz). Briefly, molecules were placed within the spatially defined framework of the simulation area, and their Brownian dynamic behavior was Procyanidin B2 simulated. At regular intervals (0.1 ms), all mobile molecules undergo a diffusive step in a random direction by a distance calculated from Ficks law. The membrane of the cylindrical stereocilia was modeled as a two-dimensional surface of area and shape equivalent to an unwrapped cylinder 10 m long and 330 nm in diameter. Longitudinal boundaries are periodic, i.e., molecules that leave the simulated area on the left are placed back in on the right. The narrow ankle region near the base of stereocilia was modeled by using two rectangular exclusion boxes to simulate the narrow constriction (see Fig. 2and (including the fluorescence puncta, arrows in and to the quantification of the data obtained from the output of the simulation using diffusion rates of 0.01 m2/s and 0.005 m2/s and stereocilia length of 10 m. Scale bars: were 191.8 19.0 (= 7) for the stereocilia membrane, 89.8 4.1 (= 5) for the region of the apical membrane around the base of the stereocilia and over the cuticular plate, and 57.9 2.5 (= 5) for the nearby background. When normalized for the background, the fluorescence over stereocilia was 133.9 and over the apical membrane was 31.9. Considering that the measured fluorescence intensity over the stereocilia corresponds to the integrated fluorescence of a series of 300-nm-diameter cylinders separated by 60 nm interstereociliary separations (estimated from electron microscopy images; data not shown), we estimate that the relative fluorescence intensity for the unwrapped stereocilia membrane is 51.2. This corresponds to 1 1.6 times the fluorescence of the apical membrane measured over the cuticular plate region. Open in a separate window Figure 1. PMCA2 distribution in the hair cell apical membrane. also shows the steady-state uniform distribution of PMCA2. The values were background subtracted and normalized to the peak value..