Cell. 2010 May 28;141(5):786-98.
Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing.
Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, Fujiwara I, Bird JE, Riazuddin S, Riazuddin S, Ahmed ZM, Hinshaw JE, Sellers J, Bartles JR, Hammer JA 3rd, Richardson GP, Griffith AJ, Frolenkov GI, Friedman TB.Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA.
Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets but distinct from bundles formed by espin, an actin crosslinker in stereocilia. We generated mutant Triobp mice (Triobp(Deltaex8/Deltaex8)) that are profoundly deaf. Stereocilia of Triobp(Deltaex8/Deltaex8) mice develop normally but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere. Copyright 2010 Elsevier Inc. All rights reserved.
When I was reading the paper above, I noticed an experiment is really interesting.
Two-Dimensional Rafts of F-actin
Two-dimensional (2D) rafts of actin filaments were formed using a modification of a technique (Taylor and Taylor, 1994; Volkmann et al., 2001) developed for decorating actin filaments with a myosin S1 fragment (Moore et al., 1970). Briefly, a 3:7 w/w lipid solution (total 1 mg/ml) of 1,2-dilaurylphosphatidylcholine (DLPT, Avanti Polar Lipids, Inc.) and didodecyldimethylammonium bromide (DDDMA, Fluka) in chloroform was spread on the surface of the polymerization buffer that contained 20mMNa2HPO4, 50mMKCl, 1mMATP, 2mM MgCl2, 1 mM EGTA, 1 mM DTT in a 20 ml Teflon well and incubated for 1 hr at 4C. G-actin was added to a solution of purified GFP-TRIOBP-4-FLAG or purified espin 3A or to a solution without an actin crosslinker. Using a glass pipette, this G-actin solution was mixed and injected into the polymerization buffer in a Teflon well and incubated for 14–16 hr at 4C. 2D-actin paracrystalline arrays formed underneath the monolayer lipid membrane (Langmuir-Blodgett film). The film was transferred to 400-mesh carbon-coated copper grids, washed with polymerization buffer, and negatively stained with 2% aqueous uranyl acetate. TEM images were taken at 100 keV with 35,000 magnification and 0.5 mm defocus (Philips CM120, Digital Micrograph). To measure the distance between actin filaments in the bundles, image analyses were performed as
described (Volkmann et al., 2001). Briefly, images were processed using a fast Fourier transform (FFT) filter to detect spatial periodicity (Metamorph, Molecular Devices). Line scan intensity was measured across the filtered image of the bundles and then fit to a sum of Gaussian distributions using the maximal likelihood method. The distance between the peaks of these distributions represents the distance between the centers of adjacent actin filaments.
This 2D rafts of F-actin was initially introduced in 1994, by Kenneth and Dianne
Biophys J. 1994 Nov;67(5):1976-83.
Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.
Taylor KA, Taylor DW.Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.
This is an assay I have never encountered before. Very interesting!
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