Tropomyosin isoforms differentially tune actin filament length and disassembly

Figures

• 

  FIGURE 1:. TPMs restrict Cof1 binding to the pointed end and block severing. OG-labeled actin filaments were preassembled in the presence or absence of the indicated human TPM isoform (2 µM). Then, excess actin monomers were flowed out and human Cy3-Cof1 (150 nM) was flowed in. (A) Representative time points and kymographs from TIRF microscopy movies with and without Tpm3.1. The pointed end of each filament is indicated by white arrowheads. For representative time points from TIRF reactions containing other TPM isoforms, see Supplemental Figure S1. Scale bars: 5 μm. (B) From time-lapse imaging (as in A and Supplemental Figure S1), severing rates were measured (n = 120, from three independent experiments). The Kd of each TPM isoform for actin filament binding (as determined by Janco et al., 2016) is indicated above the bar graph. (C) Accumulation of Cy3-Cof1 fluorescence on actin filaments in the absence (purple curves) or presence (teal curves) of Tpm3.1. (D) Distributions of time intervals between first appearance of a Cy3-Cof1 spot on an actin filament and severing event near that Cy3-Cof1 spot. The box-and-whisker plots represent minimum, median, and maximum values for each condition.…

• 

  FIGURE 2:. TPM isoform-specific protection of actin filaments from the collaborative disassembly effects of Cor1B, Cof1, and AIP1. (A) OG-actin filaments were first assembled in the presence or absence of 2 µM Tpm3.1, then the indicated concentrations of Cor1B, Cy3-Cof, and AIP1 were flowed in. Shown are representative time points and kymographs from TIRF imaging. Scale bars: 5 μm. (B) Kymographs from movies, as in A, but in the presence of different TPM isoforms (concentrations indicated). (C) Disassembly rates of actin filaments (OG labeled) in TIRF reactions measured by monitoring the decrease in OG-actin fluorescence over time (n = 60 filaments, pooled from three independent experiments). Filaments were assembled in the presence of different TPM isoforms (2 µM unless otherwise indicated), and disassembly was induced at time zero by washing out actin monomers and flowing in 150 nM Cof1, 15 nM Cor1B, and 15 nM AIP1.

• 

  FIGURE 3:. TPM decoration permits Cor1B binding but inhibits Cof1 recruitment. (A) Representative time points from TIRF microscopy imaging of Cor1B-SNAP649 binding to preassembled OG-actin filaments that were either undecorated (top) or decorated with 2 µM Tpm3.1 (bottom). (B) Accumulation of Cor1B-SNAP649 fluorescence on individial OG-actin filaments (n = 45, pooled from three independent experiments) decorated by 2 µM of the indicated TPM isoform. (C) Representative time points and kymographs from TIRF microscopy imaging, in which OG-labeled actin filaments were assembled in the presence or absence of Tpm3.1 (2 µM), and then the indicated concentrations of Cy3-Cof1 and Cor1B-SNAP-649 were flowed in. (D) Accumulation over time of Cy3-Cof1 fluorescence on actin filaments in the presence or absence of Tpm3.1 (2 µM). The average fluorescence signal is shown (n = 45 filaments, pooled from three independent experiments). Scale bars in all panels: 5 μm.

• 

  FIGURE 4:. TPM isoforms differentially tune the length of actin filaments undergoing simultaneous assembly and turnover. (A) Example of actin filaments being elongated by the formin mDia1 (1 nM) and profilin (PFN; 5 µM), while simultaneously being disassembled by Cy3-Cof1, Cor1B, and AIP1 (150, 15, and 15 nM, respectively) in the presence or absence of different TPM isoforms (2 µM unless otherwise indicated). (B) Kymographs of filaments from TIRF movies as shown in A. (C) Effects of different TPM isoforms on filament severing rate by the three-component system (Cy3-Cof1, Cor1B, and AIP1). Severing rates were obtained from kymographs as in B (n = 80 filaments per condition). (D) Filament barbed-end elongation rates, quantified from reactions as in A containing mDia1, profilin, Cy3-Cof1, Cor1B, AIP1, and different TPM isoforms (n = 80 filaments per condition; su = subunit). (E) Average length of filaments measured 15 min after reactions were initiated. (F) Average length of filaments measured 10 min after reactions were initiated. CCA indicates reactions contain Cor1B, Cof1, and AIP1. Statistical significance from the control condition (No TPM) was determined using one-way ANOVA. ****, p <…