Thin filament length regulation in striated muscle sarcomeres: pointed-end dynamics go beyond a nebulin ruler

Abstract

The actin (thin) filaments in striated muscle are highly regulated and precisely specified in length to optimally overlap with the myosin (thick) filaments for efficient myofibril contraction. Here, we review and critically discuss recent evidence for how thin filament lengths are controlled in vertebrate skeletal, vertebrate cardiac, and invertebrate (arthropod) sarcomeres. Regulation of actin polymerization dynamics at the slow-growing (pointed) ends by the capping protein tropomodulin provides a unified explanation for how thin filament lengths are physiologically optimized in all three muscle types. Nebulin, a large protein thought to specify thin filament lengths in vertebrate skeletal muscle through a ruler mechanism, may not control pointed-end actin dynamics directly, but instead may stabilize a large core region of the thin filament. We suggest that this stabilizing function for nebulin modifies the lengths primarily specified by pointed-end actin dynamics to generate uniform filament lengths in vertebrate skeletal muscle. We suggest that nebulette, a small homolog of nebulin, may stabilize a correspondingly shorter core region and allow individual thin filament lengths to vary according to working sarcomere lengths in vertebrate cardiac muscle. We present a unified model for thin filament length regulation where these two mechanisms cooperate to tailor thin filament lengths for specific contractile environments in diverse muscles.

Figure 1. Thin filament organization in striated sarcomeres

(A–C) Diagrams of vertebrate skeletal (A), vertebrate cardiac (B) and invertebrate (C) striated muscle sarcomeres showing the location of actin-binding proteins and the organization of thin and thick filaments. Elastic titin filaments (gray) extend an entire half-sarcomere from Z-line (Z) to M-line (M) to form a stable, yet flexible myofibril scaffold. Actin (thin) filaments (green) are precisely aligned with their fast-growing (barbed, B) ends at the Z-line where they are capped by capZ (cyan) and crosslinked by α-actinin (blue) to thin filaments in adjacent sarcomeres. The non-crosslinked (free) portions of the thin filaments extend ~1 µm away from the Z-line, overlap with the 1.6 µm bipolar myosin (thick) filaments (purple), and their slow-growing (pointed, P) ends are capped by tropomodulin (Tmod, red). Tropomyosin/troponin (TM/Tn) complexes copolymerize along the entire free portions of the thin filaments where they regulate myosin motor binding and bind Tmod at the pointed filament end (not shown). Nebulin (gold, A) is anchored at the Z-line and extends ~1 µm along skeletal muscle thin filaments. Nebulette (gold, B), a nebulin homolog, extends ~150 nm along cardiac muscle thin filaments. Invertebrate (arthropod) muscles (C, shown at a reduced scale) have titin-like proteins that connect thick filaments to Z-lines (gray) and do not have nebulin homologs. Thin and thick filaments elongate to maintain overlap during sarcomere growth. (D) Thin filaments (green, phalloidin) are readily observed by light microscopy. Barbed (B) and pointed (P) ends are visualized using α-actinin (blue) and Tmod (red) antibodies, respectively. A gap in phalloidin staining (H-zone, H) indicates that thin filaments are regulated in length. Narrow Tmod striations indicate that thin filament lengths are uniform. Narrow α-actinin striations indicate that thin filament barbed ends are precisely aligned at Z-lines. The distance between Tmod and associated Z-lines (L) is equal to thin filament length [81].

Figure 2. Models for determining thin filament lengths

A, ‘Static’ ruler mechanism: nebulin (red line) extends along each thin filament, binds actin monomers (green circles), and determines where Tmod (blue trapezoid) is located. In this idealized model, thin filaments remain capped for their entire lifetime and there are no actin dynamics. Except for measurement error, the distance from the Z line for Tmod (L_Tmod) is equal to and specified by the length specified by nebulin (L_nebulin). Individual thin filament lengths do not vary. B–D, Dynamic ruler mechanisms. B, ‘Cap Locator’ mechanism: dynamic capping by Tmod, recruited to L_nebulin by nebulin, allows thin filament lengths to increase or decrease. L_Tmod is equal to and specified by L_nebulin. Individual thin filament lengths vary depending on pointed end dynamics. Sub-stoichiometric nebulin amounts in cardiac muscle reduce how much Tmod is recruited to L_nebulin, resulting in a broader length distribution. C, ‘Progressive Capping’ mechanism: dynamic Tmod capping progressively slows elongation from pointed ends up to a maximum thin filament length equal to and specified by L_nebulin. However, L_Tmod is less than L_nebulin because dynamic Tmod caps at shorter lengths. Individual thin filament lengths vary depending on pointed end dynamics. D, ‘Stabilizing’ mechanism: dynamic Tmod capping allows pointed ends to extend beyond L_nebulin, yet nebulin prevents filament shortening by stabilizing the thin filament, thereby specifying a minimum length equal to L_nebulin. L_Tmod is greater than L_nebulin because dynamic Tmod caps at longer lengths. Individual thin filament lengths vary depending on pointed end dynamics. E, A Pointed-end regulation (‘Composite’) mechanism is similar to the stabilizing mechanism except that thin filament pointed end dynamics are regulated independently of nebulin in different muscles to control the length of the pointed-end extensions (L_ext). L_Tmod is specified by L_nebulin and L_ext in each muscle. Individual thin filament lengths vary for each muscle depending on pointed end dynamics which may account for L_ext in different muscles. For clarity, only one strand of actin subunits in the filament is shown.