Leiomodin 2 is a processive pointed-end elongator of actin filaments

Figures

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  Fig. 1. Lmod2 drives processive PE polymerization. A Domain diagrams of human Lmod2 wild-type, mutants and deletions used in this study. Tmod1 domain diagram is shown for comparison. TPMBS, Tpm-binding site; ABS1, actin-binding site-1; DBS, D-loop–binding site; LRR, leucine-rich repeat domain; PRD, proline-rich domain; WH2, WASP homology-2 domain. Mutated amino acids are indicated. While the N-terminal region of Tmod1’s ABS1 shows partial conservation in Lmod2, the C-terminal site does not, as indicated by the gradient coloring. In PRD*, all proline residues in the ranges 427–435 and 442–450 were replaced with glycines. B Experimental strategy: filaments nucleated from coverslip-anchored Lmod2 and 1 µM red G-actin (15% Alexa-568), elongating from 2 µM green G-actin (15% Alexa-488) ± 25 nM capping protein (CP). C, D Representative kymographs of filaments elongating under these conditions without (Movie S2) or with 25 nM CP (Movies S3–S4). E Experimental strategy: filaments nucleated from coverslip-anchored Lmod2 and 1 µM green G-actin (15% Alexa-488), transiently exposed to 10 nM 549-CP (red), and then elongating from 2 µM unlabeled G-actin. F Representative kymograph of a filament (top)…

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  Fig. 2. Direct visualization of Lmod2 during PE elongation. A Experimental strategy: a solution containing 10 nM 549-Lmod2 (red) and 2 µM G-actin (15% Alexa-488, green) was first introduced into the mf-TIRF chamber, and filaments were captured by coverslip-anchored biotin-CP. These filaments were then exposed to flow containing 2 µM G-actin (15% Alexa-488) and 25 nM unlabeled CP in TIRF buffer. B Representative kymographs of two different filaments (top) with 549-Lmod2 bound at their distal PEs (middle) elongating under these conditions (bottom, merged; Movies S7–S8). C Elongation rates (mean ± SD) of free and 549-Lmod2-bound PEs in the presence of 2 µM G-actin (15% Alexa-488). Number of filament ends analyzed: Lmod2-bound PE, 18; free PE, 8. All experiments were performed at least three independent times, and yielded similar results. Data shown are from one experiment. Source data are provided as a Source Data file.

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  Fig. 3. Lmod2-mediated PE elongation persists in the presence of profilin and tropomyosin. A Representative kymograph of an Alexa-568 (red) filament nucleated by coverslip-anchored Lmod2, elongating from 2 µM G-actin (15% Alexa-488, green) and 2 µM profilin (Movie S9). B Elongation rates (mean ± SD) of Lmod2-bound (black symbols) and free (red symbols) PEs at varying equimolar concentrations of G-actin and profilin. Black line, saturating binding curve used to estimate the plateau rate of ~1.2 subunits s−1. Number of filaments analyzed: Lmod2-bound PE (from low to high [G-actin], [profilin]: 47, 58, 65, 45, 70, 60, 68 and 56); free PE (from low to high [G-actin], [profilin]: 56, 45, 54, 18, 24, 12, 29 and 22). C Elongation rates (mean ± SD) of Lmod2-bound (orange bars) and free (green bars) PEs with 2 µM G-actin and 0 (left), 2 (middle), or 5 µM profilin (right). Number of filaments (left to right): 44, 40, 71, 25, 62, 65. Also see Fig. S4. D PE elongation rates (mean ± SD) with Lmod2 WT and PRD mutant, 2 µM G-actin, and without (left) or with 5 µM profilin (right). Number of filaments (left to right): 53, 64, 28, 80. Statistical comparison by two-sided t test against WT (ns, not…

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  Fig. 4. Role of Lmod2 domains in processive PE elongation. A Experimental strategy. Filaments were nucleated by coverslip-anchored Lmod2 using 1 µM G-actin (15% Alexa-488, green), then elongated with 2 µM unlabeled G-actin plus 25 nM CP. PE elongation rates and the time-dependent survival fraction of Lmod2-bound filaments were measured. B Representative time-lapse images of filaments (green) detaching from surface-anchored Lmod2 WT (top) and Lmod2ΔWH2 (bottom). Scale bar, 5 µm. C PE elongation rates of filaments nucleated by Lmod2 WT and mutants (mean ± SD). Numbers of filaments analyzed: WT, 53; ΔWH2, 23; DCM*, 25; ABS1*, 80. Statistical comparisons were performed against WT using two-sided Student’s t-test for ABS1* and Welch’s t-test for ΔWH2 and DCM* (ns, p ≥ 0.05; p-values: WT vs. ABS1*, 0.07; WT vs. ΔWH2, 6.18 × 10−19; WT vs. DCM*, 1.09 × 10−18). D Survival fraction of Lmod2-bound filaments over time. Experimental data (symbols) were fit to single-exponential decays (lines) to obtain dissociation rate constants. Numbers of filaments analyzed: WT, 140; ΔWH2, 112; DCM*, 128; ABS1*, 175. E Dissociation rate constants of filaments nucleated by Lmod2 WT and mutants measured from the…

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  Fig. 5. The working model for the role of Lmod2-mediated PE elongation in myofibrillogenesis. (i) A short nascent thin filament is nucleated spontaneously or by nucleators such as CapZ. Following nucleation, the filament remains capped at its BE by CapZ and is anchored at the Z-disc via alpha-actinin. The filament PE gets capped by Tmod1. (ii) Lmod2 competes with Tmod1 for PE binding. (iii) Lmod2 processively elongates the actin filament from its PE in the presence of profilin-bound G-actin while protecting it from Tmod1. (iv) In a mature thin filament, precise length is maintained by the dynamic control of polymerization via Lmod2, capping via Tmod, and depolymerization at the PE. Depolymerization is not shown.