Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Zhang, Duan Sun; Piazza, Valeria; Perrin, Benjamin J.; Rzadzinska, Agnieszka K.; Poczatek, J. Collin; Wang, Mei; Prosser, Haydn M.; Ervasti, James M.; Corey, David P.; Lechène, C.

doi:10.1038/nature10745

Cited by 202 publications

(252 citation statements)

References 16 publications

Supporting

Mentioning

235

Contrasting

Unclassified

Order By: Relevance

“…4) by capping the barbed ends of actin filaments, a role proposed for twinfilin 2 during early stereocilia growth (10). Although the measured turnover of actin in mature stereocilia is very slow, possibly to ensure that the staircase structure established during development persists through adult life, there is a region of relatively high actin turnover at the tip of each stereocilium (37). Turnover in this region may be regulated by Eps8L2, possibly in response to extrinsic physiological signals, as indicated by its molecular structure (17).…”

Section: Loss Of Eps8l2 Is Associated With Severe Progressive Hearingmentioning

confidence: 99%

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Furness

Johnson

Manor

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 nullmutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.deafness | sensory system | ion channel H earing and balance depend on the transduction of mechanical stimuli into electrical signals. Transduction involves activation of mechanically gated ion channels near the tips of the stereocilia, specialized microvilli that form the hair bundles that project from the surface of sensory hair cells (1). Stereocilia have a cytoskeletal core composed of tightly packed, cross-linked, and uniformly polarized actin filaments (2, 3). Stereociliary length is regulated to ensure the characteristic staircase-like structure of each bundle, whose overall size and shape depends on location along the sensory organ (4). In the mammalian cochlea, hair bundles usually include three rows of stereocilia coupled by several types of extracellular links (2, 5). The embryonic and postnatal development of the bundle involves elongation and thickening of stereocilia, as well as elimination of redundant stereocilia (4, 5).In the adult cochlea, the height of stereocilia within each row is similar, not only within a single hair bundle but also between the bundles of adjacent hair cells, indicating a sophisticated level of control over growth (5, 6). Stereociliary growth and maintenance involves actin-binding proteins such as espin (7,8), plastin (9), twinfilin 2 (10), gelsolin (11), and unconventional myosin motors including myosin XVa (12) and myosin IIIa (13). Currently, we do not have a complete molecular understanding of hair bundle structure or how its growth and maintenance are controlled. Recently, we showed that epidermal growth factor receptor pathway substrate 8 (Eps8) (14) is located ...

show abstract

Section: Loss Of Eps8l2 Is Associated With Severe Progressive Hearingmentioning

confidence: 99%

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Furness

Johnson

Manor

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This method can be used to visualize the distribution of isotopically labeled metabolic reporters, such as 15 N-labeled amino acids or nucleotides, and has been employed elegantly in previous work by others to track protein turnover within the tips of stereocilia in the inner ear and to visualize cardiomyocyte cell turnover. [26][27][28] These impressive results are due in part to the ability of SIMS to achieve parts-per-billion sensitivity with a dynamic range of 10 5 . 27,29,30 As noted, MIBI is capable of lateral (x,y) resolution comparable to light microscopy, with sensitivity that appears to be Figure 1 Comparison of overlap potential between fluorophores and metal tag labels.…”

Section: Methodsmentioning

confidence: 99%

“…[26][27][28] These impressive results are due in part to the ability of SIMS to achieve parts-per-billion sensitivity with a dynamic range of 10 5 . 27,29,30 As noted, MIBI is capable of lateral (x,y) resolution comparable to light microscopy, with sensitivity that appears to be Figure 1 Comparison of overlap potential between fluorophores and metal tag labels. The top panel (a) indicates the spectra of commonly used fluorophores emitting in the visible range.…”

Section: Methodsmentioning

confidence: 99%

Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

Levenson

Borowsky

Angelo

2015

Laboratory Investigation

View full text Add to dashboard Cite

The role of immunohistochemistry (IHC) in the management of cancer has expanded to provide improved diagnostic classification, as well as guidance on disease prognosis, therapy, and relapse. These new tasks require evaluation of an increasing number of protein targets; however, conventional multiplexing, usually achieved using serial tissue sections stained for a single analyte per slide, can exhaust small biopsy specimens, complicate slide-to-slide protein expression correlation, and leave insufficient material for additional molecular assays. A new approach, mass spectrometry immunohistochemistry (MSIHC), compatible with high levels of target multiplexing and suitable for use on formalin-fixed, paraffin-embedded samples can circumvent many of these issues. The strategy employs antibodies that are labeled with elemental mass tags, such as isotopically pure lanthanides not typically found in biological specimens, rather than with typical fluorophores or chromogens. The metal-labeled antibodies are then detected in tissue using lasers or ion beams to liberate the tags for subsequent mass spectrometry detection. Within a given multiplexed IHC panel, the metal labels are selected so that their respective masses do not overlap. More than 30 antibodies have been imaged simultaneously, and up to 100 antibodies could potentially be detected at once if the full available mass spectrum is deployed. MSIHC has a number of advantages over conventional IHC techniques. Background due to autofluorescence is absent and the dynamic range is 10 5 , exceeding immunofluorescence and chromogenic IHC by 100-fold and 1000-fold, respectively. Detection of labeled primary antibodies improves assay linearity over both chromogenic and fluorescent IHC. Multiplexed mass-tagged antibodies incubated simultaneously with tissue do not appear to cross-interfere, and because the mass tags do not degrade, samples are stable indefinitely. The imaging resolution of multiplexed ion-beam imaging can be better than light microscopy. With appropriate instrumentation, MSIHC has the potential to transform research and clinical pathology practice.

show abstract

“…Together, our results imply that these myosins operate during the early and most dynamic phase of hair bundle growth, during which microvilli are driven to differentiate into growing stereocilia or to regress. It is tempting to hypothesize that class III myosins contribute to the transition from a highly dynamic mode of stereocilia growth, which might involve an actin treadmill as suggested earlier (Rzadzinska et al, 2004), to a mode that seems not to depend on actin treadmill and is much more stable (Zhang et al, 2012;Narayanan et al, 2015;Drummond et al, 2015), at least in mice (Hwang et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

Lelli¹,

Michel²,

Monvel³

et al. 2016

J Gen Physiol

View full text Add to dashboard Cite

show abstract

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Cited by 202 publications

References 16 publications

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

Contact Info

Product

Resources

About