TMSB4X Actin Buffering Stoichiometry Hair vs Supporting Cells
Cell-type-resolved measurement of the actin-monomer buffer TMSB4X (β-thymosin) vs total actin (ACTG1 protein group) in E15 chick utricle. From Zhu 2019 eLife Figure 2C, 20-cell pool samples (mean ± SEM).
This is a load-bearing measurement for h09: TMSB4X is the WH2-class actin sequestration protein that directly competes with synthetic WH2-peptide hydrogels for the G-actin pool in hair cells.
Verbatim measurement
| Protein | FM1-43 high (hair cell) riBAQ | FM1-43 low (supporting cell) riBAQ | Fold (low/high) | Source |
|---|---|---|---|---|
| ACTG1 (total actin protein group) | 0.043 ± 0.001 | 0.060 ± 0.005 | 1.4× | Zhu 2019 Fig 2C |
| TMSB4X | 0.006 ± 0.002 | 0.056 ± 0.012 | 9.3× | Zhu 2019 Fig 2C |
riBAQ = relative iBAQ = molar fraction within the cell’s measured proteome.
Statistical confidence (verbatim Zhu 2019 Table 1, p.6)
Mixed-effects model with random intercept for samples (lmerTest 3.1-0):
| 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 |
Key reads:
- TMSB4X drops significantly between supporting cells and hair cells (p < 0.001).
- Hair-cell TMSB4X ≪ hair-cell actin (p = 0.001) — actin is in excess.
- Supporting-cell TMSB4X = supporting-cell actin (p = 0.66) — equimolar within error.
Mechanistic implication (paper’s argument)
1:1 binding of TMSB4X to G-actin is established (Goldschmidt-Clermont 1992).
- Supporting cells: TMSB4X / ACTG1 ≈ 0.93 → essentially all G-actin sequestered by TMSB4X.
- Hair cells: TMSB4X / ACTG1 ≈ 0.14 → ~86% of total actin is NOT TMSB4X-bound (split between F-actin polymer and free monomer).
Interpretation: developmental down-regulation of TMSB4X releases sequestered actin monomers, providing the substrate for the rapid stereocilia bundle assembly observed when hair cells differentiate without upregulating actin gene transcription.
Caveats
- One of the 5 TMSB4X peptides identified is shared with paralog TMSB15B (Zhu 2019 Fig 1—supplement 2). gEAR mouse data show Tmsb4x dominates over Tmsb10/Tmsb15 in inner ear → assumption holds in mouse, inferred in chick.
- Total iBAQ scales non-linearly with cell number → absolute riBAQ values are lower bounds; relative comparisons (the load-bearing claim) are robust.
- Membrane-protein under-detection means actin’s relative fraction may be slightly inflated in this dataset compared to a complete proteome.
Use in h09 modeling
For the h09 WH2-peptide hydrogel hypothesis:
- Free G-actin pool target. In hair cells, ~86% of actin is not TMSB4X-bound. After subtracting F-actin (the bulk in stereocilia), the free G-actin available for synthetic WH2-peptide binding is the part not in F-actin and not in TMSB4X. See Recipe — TMSB4X-Buffered Free G-actin Pool Estimation.
- Competition baseline. Endogenous β-thymosin-class binder concentration = ~0.6% of cellular protein in hair cells → a synthetic WH2 peptide must achieve effective concentration above this to meaningfully shift the equilibrium.
- Off-target. In supporting cells, TMSB4X is at near-equimolar with actin → if hydrogel diffuses to supporting cells (ototopical delivery is non-specific), it would compete with much higher local TMSB4X concentration. Supporting cells are not the therapeutic target compartment, but this is a sink term.
Connections
[source]2026-04-23-krey-2019-stereocilia-proteomics-elife[part-of]actin-kinetics[supports]TMSB4X Drop During Hair Cell Differentiation[supports]Recipe — TMSB4X-Buffered Free G-actin Pool Estimation[see-also]Recipe — Profilin Thymosin-β4 Monomer Pool Partitioning[see-also]Hair Cell Biophysical Reference Table[applies]h09 hub