Large Membrane Domains in Hair Bundles Specify Spatially Constricted Radixin Activation

Abstract

The plasma membrane of vertebrate hair bundles interacts intimately with the bundle cytoskeleton to support mechanotransduction and homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular bundles. While the bundle glycerophospholipids and acyl chains resemble those of other endomembranes, bundle ceramide and sphingomyelin nearly exclusively contain short-chain, saturated acyl chains. Confocal imaging of isolated bullfrog vestibular hair cells shows that the bundle membrane segregates spatially into at least three large structural and functional domains. One membrane domain, including the stereocilia basal tapers and ~1 μm of the shaft, the location of the ankle links, is enriched in the lipid phosphatase PTPRQ (protein tyrosine phosphatase Q) and polysialylated gangliosides. The taper domain forms a sharp boundary with the shaft domain, which contains the plasma-membrane Ca2+-ATPase PMCA2 and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2); moreover, a tip domain has elevated levels of cholesterol, PMCA2, and PI(4,5)P2. Protein mass spectrometry shows that bundles from chick vestibular hair cells contain a complete set of proteins that transport, synthesize, and degrade PI(4,5)P2. The membrane domains have functional significance; radixin, essential for hair-bundle stability, is activated at the taper-shaft boundary in a PI(4,5)P2-dependent manner, allowing assembly of protein complexes at that site. Membrane domains within stereocilia thus define regions within hair bundles that allow compartmentalization of Ca2+ extrusion and assembly of protein complexes at discrete locations.

Unconventional Mechanics of Lipid Membranes: A Potential Role for Mechanotransduction of Hair Cell Stereocilia

“…First, the morphology suggests that the upper tip-link insertion is kept rigid by a plaque containing a variety of important molecules, such as harmonin and whirlin (1), whereas membrane tenting is often observed by electron microscopy on the lower insertion site (9,(16)(17)(18). Second, electron microscopy studies have confirmed the membrane-cytoskeleton connection along the stereocilia, and more recent experiments even identified proteins such as radixin and myosin IIIa as potential cross-linker molecules for this connection (19,20). Thus, the assumption regarding the membrane-skeleton interaction in both the The red region could be tightly coupled to the stereocilia cytoskeleton (i.e., a bundle of actin filaments) through a cross-linker; blue indicates the membrane tented region into which the tip link inserts.…”

Stiffened lipid platforms at molecular force foci

“…Thus, this compliance arises either from the channel's own anchor to cytoskeleton or the channel's surrounding membrane (35,36), which may also be anchored. Lipids are not evenly distributed in the stereociliary membrane (37). Filipin, a cholesterolbinding fluorescent antibiotic, stains the very tip of the stereocilia, where the transduction channels reside (37) (Fig.…”

“…Lipids are not evenly distributed in the stereociliary membrane (37). Filipin, a cholesterolbinding fluorescent antibiotic, stains the very tip of the stereocilia, where the transduction channels reside (37) (Fig. 1A).…”

Apart from its basic function the plasma membrane Ca2+ATPase regulates Ca2+ signaling by controlling phosphatidylinositol-4,5-bisphosphate levels

“…If we take the assumption that about 5000-10,000 PtdIns(4,5)P 2 molecules are in a square micron of the plasma membrane we might assume that most of them are sequestered by the large amount of PMCA in the stereocilia. In good accordance with this hypothesis, recent studies have demonstrated that PtdIns(4,5)P 2 is concentrated at the tips of stereocilia together with the PMCA2 protein (Zhao et al, 2012). Further experiments have demonstrated that a specific asymmetric distribution of PtdIns(4,5)P 2 (Hirono et al, 2004) and PMCA2 (Kozel et al, 1998) is essential for the appropriate function of hair cells, underlining the importance of a putative CPPP in hearing.…”

“…We suggest that buffering of PtdIns(4,5)P 2 by the PMCAs might have a substantial impact on PtdIns(4,5)P 2 signaling when a large amount of pump is being expressed at discrete locations, like the dendritic spines or the stereocilia of hair cells (Burette et al, 2010;Zhao et al, 2012). We must emphasize, however, that even a small amount of active PMCA could be protective if it is located near the PtdIns(4,5)P 2 pool.…”