Technical Reference #1819

1819.

Differential Localization and Dynamics of Class I Myosins in the Enterocyte Microvillus Andrew E. Benesh; Rajalakshmi Nambiar; Russell E. McConnell; Suli Mao; David L. Tabb; and Matthew J. Tyska, Vanderbilt University School of Medicine, Molecular Biology of the Cell, 21(1819), (2010)
Link To Paper

Abstract:
Epithelial cells lining the intestinal tract build an apical array of microvilli known as the brush border. Each microvillus is a cylindrical membrane protrusion that is linked to a supporting actin bundle by myosin-1a (Myo1a). Mice lacking Myo1a demonstrate no overt physiological symptoms suggesting that other myosins may compensate for the loss of Myo1a in these animals. To investigate changes in the microvillar myosin population that may limit the Myo1a KO phenotype we performed proteomic analysis on WT and Myo1a KO brush borders. These studies revealed that WT brush borders also contain the short-tailed class I myosin myosin-1d (Myo1d). Myo1d localizes to the terminal web and striking puncta at the tips of microvilli. In the absence of Myo1a Myo1d peptide counts increase twofold; this motor also redistributes along the length of microvilli into compartments normally occupied by Myo1a. FRAP studies demonstrate that Myo1a is less dynamic than Myo1d providing a mechanistic explanation for the observed differential localization. These data suggest that Myo1d may be the primary compensating class I myosin in the Myo1a KO model; they also suggest that dynamics govern the localization and function of different yet closely related myosins that target common actin structures.

Materials & Methods:
FRAP was performed as previously described (Tyska and Mooseker 2002). Briefly LLC-PK1-CL4 cells stably expressing EGFP-Myo1d or EGFP-Myo1a were grown to confluency on MatTek (Ashland MA) 35-mm glass-bottomed dishes. Before imaging complete medium was exchanged for 25 mM HEPESbuffered DMEM lacking phenol red and mineral oil was layered on top to prevent medium evaporation. Cells were incubated in a WeatherStation (Precision Control Sammamish WA; http://www.precisioncontrol.net/) at 37°C and imaged using an Olympus FV-1000 laser scanning confocal microscope. A field of 105.6 105.6 m was scanned every 3 s and a region of interest (ROI) for photobleaching was set at 5 m2. Bleaching was performed with three scans at 100% transmission. Immediately after bleaching the entire field was scanned at 3-s intervals. ImageJ was used to extract the ROI integrated pixel intensities from raw data files; intensity data were then exported to a Microsoft Excel (Redmond WA) where background and t 0 intensity values were subtracted from each time point. Intensity data were normalized so that the scan immediately before photobleaching was equal to 1. Using SigmaPlot (v.10; Systat San Jose CA) recovery data were fit to the following kinetic model: IROI (t 0) A1exp k1t A2exp k2t where IROI is the ROI intensity at time t 0 is the mobile fraction and Ax is the amplitude of the exponential process with rate kx.

Microscopic Technique
Laser Scanning Confocal Microscope

Cell Type(s)
LLC-PK1-CL4 cells