Thin Filament Pointed Ends Redistribute in Response to Altered Thick Filaments

Abstract

Striated muscle generates contractile force and shortens as polar actin (thin) filaments slide past bipolar myosin (thick) filaments. Maximal force is able to be produced when thin filaments completely overlap the motor domains (heads) which occurs when the thin filament slow-growing (pointed) ends extend to the central (∼0.2 micrometer) thick filament bare zone devoid of myosin heads. The assembly of thin and thick filaments into regular contractile units (sarcomeres) is complex and highly-regulated, yet also robust; allowing diverse vertebrate and invertebrate animals to produce a variety of sarcomeric architectures with specific physiological contractile properties. Maximal thin-thick filament overlap in vivo is highly conserved among all types of striated muscle examined and highlights its importance. How thin-thick filament overlap is specified and maintained in different muscles is uncertain, but it manifests as the uniform and precise specification of thin filament lengths and involves the exchange of actin subunits at pointed ends and dynamic pointed-end capping by tropomodulin (Tmod). Here, we use CRISPR-Cas9 gene editing and fluorescence microscopy to investigate whether interactions between myosin heads and pointed ends are necessary for specifying thin filament lengths in C. elegans obliquely striated body wall muscle. To determine the position of thin filament pointed ends, we inserted the green fluorescent protein (GFP) coding sequence in-frame with the C. elegans Tmd1 gene to generate a GFP-Tmod fusion protein. Homozygous GFP-Tmod worms are indistinguishable from wildtype worms demonstrating that the GFP fusion is completely functional. In body wall muscle, GFP-Tmod appears as regular striations that co-localize with myosin thick filaments. GFP-Tmod striations appear as single bands (singlets) or closely spaced double bands (doublets) in living worms confirming that thin-thick filament overlap is complete. In contrast, GFP-Tmod striations appear as more broadly-spaced doublets in headless-myoA transgenic worms that were designed to have an expanded (∼2 micrometer) thick filament bare zone. Our results show that thin filament pointed ends redistribute their location in response to changing the size of the thick filament bare zone. The response of pointed ends suggests that interactions with myosin heads stabilize pointed ends, increasing thin filament lengths until they fully overlap with thick filaments and permit optimal force generation.