The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments.

Abstract

Abstract. Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends.Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3/zm) and thick filament (from 4.1 to 3.3 t~m) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A-and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap illaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the 1-bands by elastic recoil, in highly stretched fibers.

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…The best supported hypothesis stems from observations that thick filament shortening 1) is not associated with changes in thick filament helical structure but 2) is associated with changes in thick filament charge , phosphorylation state , and the appearance of unattached thick filament end fragments (Levine et al, 1991b) and 3) regulatory light chain phosphorylation causes the release of similar end fragments in vitro (Levine et al, 1991a). These observations are consistent with thick filament shortening occurring via a reversible, phosphorylation dependent, disaggregation of thick filament ends.…”

Simultaneous Stretching and Contraction of Stress Fibers In Vivo

“…In regions where there was a shortening of the sarcomeres, the individual bands became shorter, whereas in regions where there was stretching, these bands became wider. However, an exception is found in Limulus telson muscle in which A-bands are shorter with ragged edges in stretched, activated muscle preparations, and this shortening is attributed to filament degradation (Levine et al, 1991). Again, this variation in band width distinguishes the behavior of these structures from the behavior of striated muscle sarcomeres.…”

Elastic Properties of Isolated Thick Filaments Measured by Nanofabricated Cantilevers

“…Second, both of the muscles we selected have been at the center of scientific interest for years, yet many questions about their ultrastructure and working mechanisms remain unanswered, questions about long-term tension maintenance in the catch state (cf. Twarog, 1967;Sobieszek, 1973;Nonomura, 1974;Siegman et al, 1997) and questions about thick filament shortening (de Villafranca and Marschhaus, 1963;Dewey et al, 1977;Levine and Kensler, 1985;Levine et al, 1991).…”