2019 Zhu — Single-cell proteomics reveals changes in expression during hair-cell development
Single-cell proteomics of E15 chick utricle: TMSB4X actin-buffer is 10× lower in hair cells than supporting cells (riBAQ 0.006 vs 0.056), releasing G-actin for stereocilia.
Citation
Zhu Y, Scheibinger M, Ellwanger DC, Krey JF, Choi D, Kelly RT, Heller S, Barr-Gillespie PG. “Single-cell proteomics reveals changes in expression during hair-cell development.” eLife 8:e50777, 2019. DOI 10.7554/eLife.50777. Published 2019-11-04. CC0 public domain.
Filename note: PDF is krey2019.pdf because Krey is corresponding-author lab. First author is Zhu.
Earlier-note correction (2026-04-25): previous version of this note attributed the paper to “Krey JF, Chatterjee P, Halford J, Cunningham CL, Perrin BJ, Bharat TAM…” — that author list was hallucinated. Verified-correct list above is taken verbatim from the locally-parsed MinerU output of the eLife PDF (~/BookLibrary/mineru-output/krey2019/krey2019/auto/krey2019.md, line 19).
TL;DR
NanoPOTS single-cell proteomics on FACS-sorted E15 chick utricle cells (1 cell ≈ 1 picoliter) revealed that the actin-monomer buffer TMSB4X is at near-equimolar concentration with actin in supporting cells (progenitors) but drops ~10-fold in hair cells. The authors propose this drop releases sequestered G-actin for stereocilia bundle assembly, explaining why hair cells build elaborate F-actin without upregulating actin gene transcription. CellTrails developmental-trajectory inference reproduces the supporting-cell → hair-cell transition from protein expression alone (30 single cells) and corroborates with scRNA-seq from 254 cells.
Numbers that matter
Cell biophysical parameters
| Parameter | Value | Method | Source (page) |
|---|---|---|---|
| FM1-43 high (hair cell) volume | 1.01 ± 0.20 pL (mean ± SD, N=9) | post-FACS volumetric | p.3 |
| FM1-43 low (supporting cell) volume | 0.67 ± 0.15 pL (N=8) | post-FACS volumetric | p.3 |
| HeLa cell volume (comparator) | 4.9 ± 1.1 pL (N=5) | same method | p.3 |
| Stereocilia per E15 utricle hair cell | 72 ± 8 (N=26 striolar + extrastriolar) | wholemount counts | p.5 |
| Total actin per E15 hair cell | ~15,000,000 molecules | ACTG1 riBAQ × volume | p.5 |
| Actin per E15 stereocilium | ~200,000 molecules | derived from 15M / 72 | p.5 |
| Actin per E20 stereocilium (Shin 2013 ref) | ~400,000 molecules | external prior | p.5 |
| Single FM1-43 high cell, unique peptides | ~200 (50% MS/MS, 50% Match) | nanoPOTS LC-MS/MS | p.4 |
| 20-cell pool FM1-43 high, unique peptides | ~2,500 (~70% MS/MS) | same | p.4 |
| Proteins detected in single FM1-43 high cell | ~60 | identification threshold | p.4 |
| Proteins detected in 20-cell FM1-43 high pool | ~600 | same | p.4 |
TMSB4X vs actin stoichiometry (load-bearing for h09)
Verbatim from Figure 2C, 20-cell pool samples (mean ± SEM):
| Protein | FM1-43 high (hair cell) riBAQ | FM1-43 low (supporting cell) riBAQ | Fold (low/high) |
|---|---|---|---|
| ACTG1 (total actin) | 0.043 ± 0.001 | 0.060 ± 0.005 | 1.4× |
| TMSB4X | 0.006 ± 0.002 | 0.056 ± 0.012 | 9.3× |
Stoichiometry consequence: TMSB4X binds actin 1:1 (Goldschmidt-Clermont 1992).
- Supporting cells: TMSB4X ≈ ACTG1 → most actin monomers sequestered
- Hair cells: TMSB4X / ACTG1 ≈ 0.14 → ~86% of actin not bound by TMSB4X
Statistical confidence (verbatim Table 1, p.6)
| Comparison | Estimate | Std. error | df | t-value | 95% CI lower | 95% CI upper | p-value |
|---|---|---|---|---|---|---|---|
| ACTG1 FM-high − ACTG1 FM-low | −0.017 | 0.009 | 22.3 | −1.830 | −0.036 | 0.002 | 0.081 |
| ACTG1 FM-high − TMSB4X FM-high | 0.036 | 0.008 | 12.0 | 4.607 | 0.019 | 0.054 | 0.001 |
| ACTG1 FM-low − TMSB4X FM-low | 0.004 | 0.008 | 12.0 | 0.451 | −0.014 | 0.021 | 0.66 |
| TMSB4X FM-high − TMSB4X FM-low | −0.050 | 0.009 | 22.3 | −5.356 | −0.069 | −0.031 | <0.001 |
Mixed-effects model with random intercept for samples, lmerTest 3.1-0 (Kuznetsova 2017).
Single-cell trajectory analysis parameters
| Parameter | Value | Source (page) |
|---|---|---|
| Single cells in proteomics trajectory | 30 | p.7 |
| Proteins/groups passing detection ≥5 cells | 75 | p.7 |
| High-variance proteins for embedding | 37 (log2 niBAQ > 1.0 + above variance fit) | p.7 |
| Manifold dimensions | 4 | Methods |
| scRNA-seq cells (post-QC) | 254 (S1–S7), of 384 starting | p.10 |
| scRNA-seq genes after filtering | 186 assay genes (182 prior + AGR3, AK1, GPX2, TMSB4X) | p.10 + Table 3 |
| Smoothing spline DOF | 4 | Methods |
| Sequencing depth | 2 × 150 bp paired-end on NextSeq 500/550 High Output | Methods |
Derived [F-actin] (calculation, NOT in paper)
Re-derived from this paper’s Numbers + standard stereocilium geometry. Flagged as derived.
| Parameter | Value | Notes |
|---|---|---|
| Mature OHC stereocilium diameter | ~200 nm | external (Tilney) |
| Mature OHC stereocilium length | ~3 μm | external (Tilney) |
| Stereocilium volume | ~9.4 × 10⁻¹⁷ L | π·r²·h |
| Total actin per stereocilium | 200,000 molecules (this paper) | p.5 |
| Local [actin] in stereocilium | ~3,500 μM (3.5 mM) total | Avogadro |
| [F-actin] assuming 90% filamentous | ~3.15 mM | derived |
| Practical working estimate | 1–5 mM [F-actin] in mature core | bracketed |
Method essentials
- Tissue: E15 Gallus gallus utricle, peeled epithelium → cells dissociated (Accutase + thermolysin).
- Sorting: FM1-43 dye loading distinguishes hair cells (high) from supporting cells (low). FACS into nanowells (single + pools of 3, 5, 20).
- Sample prep: nanoPOTS (Zhu 2018b, Nat Commun) — all protein extraction, reduction, alkylation, proteolysis (trypsin + Lys-C) inside one nanowell, robotic dispensing.
- LC-MS: 30-μm-i.d. column → Orbitrap Fusion Lumos Tribrid, data-dependent acquisition.
- ID/quant: MaxQuant + Andromeda, Match Between Runs, iBAQ (intensity-based absolute quantification, Schwanhäusser 2011); per-protein iBAQ normalized as riBAQ (relative molar fraction) and niBAQ (log2 batch-corrected for trajectory).
- Trajectory inference: CellTrails R package (Bioconductor 10.18129/B9.bioc.CellTrails), spectral embedding into 4D manifold, pseudotime via diffusion-pseudotime style approach.
- scRNA-seq: Smart-seq2 (Picelli 2014) on 384 FACS-sorted cells, paired-end 2×150 bp NextSeq, STAR + RSEM, SCnorm normalization.
- Statistics: mixed-effects model (lmerTest 3.1-0); Benjamini-Hochberg FDR for volcano plot.
- Antibody validation: anti-TMSB4X validated by knockdown in two prior papers (Zhou 2013, Li 2018); chicken TMSB4X = mouse/human within 2 amino acids of 44 (95.5% identity).
Data deposit: ProteomeXchange PXD014256 (PRIDE).
Limitations
- Only 30 single cells in proteomics trajectory — insufficient to recover the TrS / TrES / TrES* triple-branching seen in the 254-cell scRNA-seq.
- Membrane proteins systematically underrepresented (general shotgun-MS limitation; Kar 2017). Only ATP1A1, SLC17A8, ATP2B2 detected. STRC absent — neither in proteomics nor in the 186-gene scRNA-seq panel (Table 3, verified). Chick utricle ≠ mammalian cochlear OHC; STRC’s HTC role is mammalian-OHC-specific.
- ACTB vs ACTG1 cannot be discriminated by mass spec here (only one isoform-distinguishing peptide detected); only scRNA-seq separates them.
- Total iBAQ scaled non-linearly with cell number (Figure 1F) → significant protein loss to nanowell-surface adsorption. Absolute counts are lower bounds.
- TMSB4X peptides covered 75% of the ~5 kDa protein (5 peptides). One of the 5 peptides is shared with paralog TMSB15B (Figure 1—supplement 2). Functional argument hinges on chick Tmsb4x vs Tmsb10/Tmsb15 dominance — supported by gEAR mouse data, not directly measured here.
Relevance to STRC
h09 (Hydrogel) — load-bearing biology
This paper supplies the first quantitative cell-type-resolved measurement of TMSB4X:actin stoichiometry in hair cells. h09’s WH2-peptide bundling mechanism is functionally analogous to TMSB4X’s WH2-class actin-monomer-binding role.
Direct implications for h09 modeling:
- Hair-cell free G-actin pool exists — TMSB4X consumes ~14% of actin, leaving ~86% as either free monomer or in F-actin. Working competition target for synthetic WH2 peptide is the unsequestered pool.
- Supporting cells are NOT the therapeutic target compartment in h09 (the bundle is in hair cells), but the supporting-cell TMSB4X excess illustrates that endogenous β-thymosin-class binders saturate ~100% of actin when at equimolar abundance — consistent with peptide binding equilibria assumed in Recipe — Profilin Thymosin-β4 Monomer Pool Partitioning.
- STRC is NOT measured here. The placeholder STRC_NORMAL_OHC_M = 1 µM remains unsourced (audit confirmed below).
STRC absence — confirmed (carried over from 2026-04-25 audit)
- Main article body, Figure 1—source data 1 (full MaxQuant output), Figure 5—source data 1 (CellTrails scRNA-seq), Table 3 (186 trajectory genes): STRC / stereocilin / Q7RTU9 not present.
- Reason: chick utricle E15 ≠ mammalian cochlear OHC. STRC’s horizontal-top-connector role is mammalian-OHC-specific.
- Verdict: STRC_NORMAL_OHC_M = 1 µM stays an in-house placeholder. Propagate
# WARNING: no primary measurementto scripts.
h26 (Engineered Homodimer Avidity) — minor
- Establishes baseline stereocilia-protein abundance in immature hair cells. Useful for calibrating effective avidity windows, but not load-bearing.
Connections
[part-of]actin-kinetics[part-of]stereocilia-mechanics[supports]Hydrogel Phase 4d F-actin Bundling Model — provides 200,000 actin/stereocilium baseline for [F-actin] derivation[supports]TMSB4X Drop During Hair Cell Differentiation[supports]Hair Cell Biophysical Reference Table[supports]Recipe — TMSB4X-Buffered Free G-actin Pool Estimation[see-also]Recipe — Profilin Thymosin-β4 Monomer Pool Partitioning[see-also]2026-04-23-pollard-2016-actin-review-cshpb[see-also]2026-04-23-chereau-wh2-actin-pnas[applies]h09 hub[about]Stefan Heller