STRC Research

Recipe — TMSB4X-Buffered Free G-actin Pool Estimation

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Recipe — TMSB4X-Buffered Free G-actin Pool Estimation

Goal: Given a hair cell’s measured TMSB4X and total actin abundance, partition the cellular actin pool into (a) TMSB4X-sequestered G-actin, (b) F-actin in stereocilia, and (c) free G-actin available for binding by an exogenous WH2 peptide (h09 hydrogel).

When to use

  • Setting up h09 hydrogel competition kinetics (peptide vs endogenous TMSB4X for monomer pool).
  • Predicting free [G-actin] in a hair-cell compartment for any monomer-binding ligand model.
  • Sanity-checking whether a candidate WH2 peptide concentration is plausible vs the endogenous actin-buffer setpoint.

Inputs (all from primary literature, see § Sources)

SymbolMeaningDefault valueSource
riBAQ_actintotal actin molar fraction (ACTG1 group)0.043 (hair cell)Zhu 2019 Fig 2C
riBAQ_tmsb4xTMSB4X molar fraction0.006 (hair cell)Zhu 2019 Fig 2C
K_d_tmsb4xTMSB4X · G-actin Kd~2 μM (vertebrate)Goldschmidt-Clermont 1992
[actin_total]total cellular actinderived from cell volume + 15M moleculesZhu 2019 p.5
f_F_stereociliafraction of total actin that is in stereocilia F-actin90% in mature; 60–80% in E15Tilney & Tilney 1988; Zhu 2019

Procedure

Step 1 — Cellular [actin]_total

[actin]_total = N_actin / (V_cell × N_A)
            ≈ 15e6 / (1.01e-12 L × 6.022e23)
            ≈ 24.7 μM   (E15 hair cell, total actin)

For a mature OHC (smaller than E15 utricle hair cell, more compact, more stereocilia), use cell-volume-corrected total. Lacking direct OHC measurement, scale by stereocilia count and use 1–5 mM inside stereocilia while bulk cytoplasm stays in low-μM. WARNING: no primary OHC actin-copy-number measurement; OHC numbers extrapolated.

Step 2 — TMSB4X-bound vs free monomer (in hair cell)

Use Zhu 2019 ratio: TMSB4X / ACTG1 = 0.006 / 0.043 ≈ 0.14.

Per the paper’s text, TMSB4X binds 1:1. Upper bound on sequestered actin:

[TMSB4X]_total ≈ 0.14 × [actin]_total ≈ 3.5 μM    (hair cell)
[TMSB4X]_total ≈ 0.93 × [actin]_total ≈ 23 μM     (supporting cell — sink term)

Equilibrium partition between sequestered, free monomer, and F-actin:

[G_free] ≈ [G_total_cytoplasm] - [TMSB4X·G]
[TMSB4X·G] ≈ min([TMSB4X]_total, [G_total_cytoplasm])
            (since K_d ≈ 2 μM ≪ working concentrations)

For mature OHC bulk cytoplasm (NOT inside stereocilia), assume 5–15% of total actin is monomeric (Pollard 2016 review):

[G_total_cytoplasm] ≈ 0.10 × [actin]_total
[G_free] ≈ [G_total_cytoplasm] − [TMSB4X·G]

If TMSB4X exceeds G-pool → [G_free] → 0, exogenous WH2 must compete with TMSB4X. If TMSB4X is below G-pool (Zhu 2019 hair-cell case) → [G_free] > 0, exogenous WH2 has substrate.

Step 3 — F-actin in stereocilia

N_actin_per_stereocilium ≈ 200,000     (Zhu 2019, E15 utricle)
                           400,000     (Shin 2013, E20 cochlea — bracketed prior)
V_stereocilium = π · r² · h            (200 nm Ø, 3 μm = 9.4e-17 L)
[F-actin]_stereo = N / (V × N_A) ≈ 1–5 mM

Use 1–5 mM as the [F-actin] bracket inside the stereocilium core; this is NOT the bulk cytoplasmic concentration.

Step 4 — Synthetic WH2 effective concentration target

For an exogenous WH2 peptide (h09) to meaningfully sequester G-actin:

[WH2_peptide]_effective ≳ [TMSB4X]_total / (K_d_tmsb4x / K_d_WH2_actin)
                       ≈ 3.5 μM × (K_d_WH2 / 2 μM)    (hair cell)

If WH2’s Kd ≈ TMSB4X’s Kd, target is ~3 μM intracellular. If WH2’s Kd is order of magnitude weaker (~20 μM), target is ~30 μM intracellular.

Practical floor: any concentration ≪ 1 μM is unlikely to compete with the endogenous TMSB4X buffer in either cell type.

Failure modes / what to flag in code

# WARNING: STRC_NORMAL_OHC_M = 1 µM is an in-house placeholder; no primary
# measurement quantifies STRC copy number in mammalian OHC.
# WARNING: f_F_stereocilia = 0.90 is from Tilney 1988 mature-cell estimate;
# E15 immature hair cells closer to 0.60–0.80.
# WARNING: cytoplasmic G-actin / total actin partition assumed 0.10
# (Pollard 2016 generic value); not directly measured in hair cells.

Worked example — h09 ear-drop concentration cross-check

Q: At a target intracellular [WH2 peptide] = 5 μM (hair cell), is the ototopical-drop concentration in STRC h09 PKPD Window Calc reachable?

Step 1: Hair-cell [actin]_total ≈ 25 μM
Step 2: [TMSB4X]_total ≈ 3.5 μM
Step 3: [G_free] ≈ 0.10 × 25 − 3.5 ≈ −1 μM → near-zero (TMSB4X consumes whole G-pool)
        Therefore exogenous WH2 must outcompete TMSB4X for the same monomers.
Step 4: For WH2 Kd matched to TMSB4X (~2 μM), need [WH2]_intracell ≳ 3.5 μM

→ 5 μM intracellular target is plausible if Kd ≤ TMSB4X’s. If WH2 Kd is 50 μM (h09 wet-lab gate), required intracellular concentration jumps to ≳ 90 μM, which is outside the current ear-drop PKPD window. This is the same gate already noted on the h09 hub.

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