Fig 2: mDia1 and mDia3 are not required for tropomyosin recruitment to actin filaments in cells at steady state. (a) Expression of actin nucleators in BJeH cells assessed via qPCR. (b,c) mDia1 (b) and mDia3 (c) were depleted separately in BJeH cells via lentiviral-mediated delivery of shRNAmir, knock down was confirmed in 3 different passages via western blotting. (d,e) non-targeting shRNA, mDia1 KD and mDia3 KD cells were fixed and co-stained for (d) F-actin and Tpm3.1/3.2 or (e) Tpm1.6/1.7/2.1 and Tpm4.2. mDia1 KD and mDia3 KD cells made poorer cell-cell junctions (stemless arrow heads) than non-targeting shRNA control cells (stemmed arrow heads). Per group a zoomed in image of one junction is displayed on the right. (f) Partitioning of tropomyosins and actin with F-actin pellet was measured via a biochemical assay in mDia1 and mDia3 KD cells as well as non-targeting shRNA expressing cells. Graph shows data from 3 independent experiments presented as mean ± SD. (g) Protein levels of tropomyosins were measured via western blotting, normalised to a-tubulin and compared between mDia1 KD, mDia3 KD and non-targeting shRNA expressing BJeH cells. Graph shows quantification across 3 independent experiments presented as mean ± SD. **P < 0.01, *P < 0.05. Scale bar shows 10 µm unless stated otherwise.

Fig 3: Co-sedimentation of S100A6 with F-actin–tropomyosin complex. (A) Saturation of the F-actin–tropomyosin complex with S100A6 as a function S100A6:F-actin molar ratio. The experimental points were fit to hyperbolic equation. (B) Fractional saturation of F-actin with S100A6 shown as a function of unbound S100A6. The experimental points were fit to the Hill equation (Eq. 1). Conditions: 3 µM F-actin, 0.5 µM tropomyosin, 0–20 µM S100A6, 10 mM Tris, pH, 7.5, 50 mM NaCl and 1 mM CaCl2. The data are from 3 independent experiments. (C) Proteins collected in pellets (p) and supernatants (s) at saturation point, separated in SDS-PAGE (12% gel). 3 µM F-actin saturated with either 0.5 µM Tpm1.6 (p1, s1) or 0.5 µM Tpm1.8 (p2, s2) and 14 µM S100A6.

Fig 4: mDia1 and mDia3 are not required to recover Tpm1.6/1.7/2.1 and Tpm4.2 containing stress fibre structures after Lat A washout. (b) mDia1 KD, (c) mDia3 KD and (a) non-targeting shRNA transfected BJeH cells were treated with DMSO or LatA and fixed and stained for Tpm1.6/1.7/2.1 (tm311) and Tpm4.2 (d9d) at different time points after wash out (w/o). Directly after LatA treatment Tpm1.6/1.7/2.1 and Tpm4.2 co-localises with F-actin foci (arrows).

Fig 5: Interaction of S100A6 with tropomyosin isoforms. (A) Pull-down assay with the use of purified Tpm1.6 or Tpm1.8 and with CNBr-Sepharose-S100A6 affinity resin (upper panels) or CNBr-Sepharose-empty resin (lower panels). Lanes: 1—input, 2—unbound fraction, 3—last wash, 4—first wash with 150 mM NaCl, 5—last wash with 150 mM NaCl, 6—first wash with 500 mM NaCl, 7—last wash with 500 mM NaCl, 8—first wash with 1 M NaCl, 9—last wash with 1 M NaCl, 10—elution. Fractions were analyzed by SDS-PAGE (10% gel) stained with Commassie brilliant blue R250. (B) ELISA assay. The amount of S100A6 bound to Tpm was estimated using anti-S100A6 antibodies. The points represent mean absorbance values ± SD from 3 independent experiments.