Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin

Figures

• 

  FIGURE 1. (A) Schematic diagrams of bulk P‐end assembly assays. Upper: Elongation of a B‐end capped F‐actin seed occurs at the P‐end when G‐actin > 0.6 μM. Lower: When Tmod caps the P‐end of a B‐end capped F‐actin seed, actin assembly at the P‐end is blocked. Tpm enhances Tmod's P‐end capping activity. (B) An example of kinetic measurements of pyrene‐actin polymerization with CapZ‐capped F‐actin seeds (CapZ:actin = 1:100). The data show actin polymerization in the presence or absence of CapZ‐capped F‐actin seeds, Tpm (TM5NM1), Tmod1, and Tmod3 as indicated in the figure. (C) An example of P‐end capping activity analysis of Tmod based on pyrene‐actin polymerization kinetics shown in B. P‐end capping activity of Tmod1 and Tmod3 was evaluated by measuring their ability to inhibit the initial elongation rates from the P‐ends of B‐end capped F‐actin seeds. The initial elongation rates were measured from the slopes of the polymerization traces over the first 1 min. The P‐end capping efficiencies of Tmod for TM5NM1‐coated F‐actin seeds were calculated using the formula shown in C. The data show that Tmod1 and Tmod3 exhibit similar P‐end capping efficiencies in the presence of TM5NM1. Data shown…

• 

  FIGURE 2. A flow chart of the experimental steps for a pyrene‐actin polymerization assay using a total volume of 300 μL.

• 

  FIGURE 3. (A) Schematic representation of the PDMS chamber used for microfluidics‐assisted TIRF (mf‐TIRF) imaging of individual actin filaments. (B) Left: A representative field of view containing actin filaments anchored at their barbed ends on the glass coverslip. The distal pointed ends are free, and the filaments are aligned under the flow. Right: The methodology used for determining the time‐point of arrest of P‐end growth by Tmod from kymographs. Scale bar, 10 μm. (C) Schematic representation of the experimental strategy used for investigating pointed‐end capping by Tmod. A solution containing G‐Actin was introduced into a mf‐TIRF flowcell in which Biotinylated SNAP‐CapZ was anchored on the glass coverslip. Pre‐formed filaments, captured by Biotinylated‐CapZ at their barbed ends, continued to elongate at their pointed ends by polymerization of G‐Actin in the solution. A solution containing G‐Actin (10 μM) and Tmod1 (0.5 μM) (in TIRF buffer) was then flowed into the chamber, and filament elongation was monitored until each filament paused. (D) Representative kymograph showing an Alexa‐488 labelled actin filament bound to coverslip‐anchored CP at its barbed end and elongating from its…

• 

  FIGURE 4. Lmod2 Enhances Actin incorporation and dynamics at thin filament pointed ends. (A) Microinjection of Rho‐Actin in GFP (Upper) and GFP‐Lmod2 overexpressing (OE) (Lower) neonatal rat cardiomyocytes. Staining for α‐actinin marks the Z‐disk where the barbed ends of the Actin filament are located (pink arrows); pointed ends are denoted by blue arrowheads. GFP‐Lmod2 often localizes to the pointed end and along the length of the thin filament but is excluded from the Z‐disk; the non–pointed‐end localization is likely of low affinity and/or nonspecific (Tsukada et al. 2010) (Scale bar: 1 μm.) (A′) Plot profile of Rho‐Actin in cells transduced with GFP (pink) and GFP‐Lmod2 (orange). (B–D) FRAP of GFP‐cardiac Actin in rat cardiomyocytes transduced with mCherry or mCherry‐Lmod2. (B) Representative images of rat cardiomyocytes before and after photobleaching. Barbed (pink arrow) and pointed (blue arrowheads) ends of the Actin filaments. (Scale bar: 1 μm.) (C) Mean relative recovery following photobleaching over time ± SEM. (D) Mean slow and fast mobile fractions ± SEM; n = 9–15. *p < 0.05, ****p < 0.0001. Used with permission from (Pappas et al. 2015).