Hair bundles of the inner ear have a unique structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis, and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin crosslinkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Summary Aggregated alpha-synuclein inclusions are found where cell death occurs in several diseases, including Parkinson’s Disease, Dementia with Lewy Bodies and Multiple System Atrophy. However, the relationship between inclusion formation and an individual cell’s fate has been difficult to study with conventional techniques. We developed a system that allows for in vivo imaging of the same neurons over months. We show that intracerebral injection of preformed fibrils of recombinant alpha-synuclein can seed aggregation of transgenically-expressed and endogenous alpha-synuclein in neurons. Somatic inclusions undergo a stage-like maturation, with progressive compaction coinciding with decreased soluble somatic and nuclear alpha-synuclein. Mature inclusions bear the post-translational hallmarks of human Lewy pathology. Long-term imaging of inclusion-bearing neurons and neighboring neurons without inclusions demonstrates selective degeneration of inclusion-bearing cells. Our results indicate that inclusion formation is tightly correlated with cellular toxicity and that seeding may be a pathologically relevant mechanism of progressive neurodegeneration in many synucleinopathies.

The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle's longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.

Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.