W. Obermann

Molecular structure of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin

The M band of sarcomeric muscle is a highly complex structure which contributes to the maintenance of the regular lattice of thick filaments. We propose that the spatial coordination of this assembly is regulated by specific interactions of myosin filaments, the M band protein myomesin and the large carboxy‐terminal region of titin. Corresponding binding sites between these proteins were identified. Myomesin binds myosin in the central region of light meromyosin (LMM, myosin residues 1506–1674) by its unique amino‐terminal domain My1. A single titin immunoglobulin domain, m4, interacts with a myomesin fragment spanning domains My4–My6. This interaction is regulated by phosphorylation of Ser482 in the linker between myomesin domains My4 and My5. Myomesin phosphorylation at this site by cAMP‐dependent kinase and similar or identical activities in muscle extracts block the association with titin. We propose that this demonstration of a phosphorylation‐controlled interaction in the sarcomeric cytoskeleton is of potential relevance for sarcomere formation and/or turnover. It also reveals how binding affinities of modular proteins can be regulated by modifications of inter‐domain linkers.

Characterization of muscle filamin isoforms suggests a possible role of γ-filamin/ABP-L in sarcomeric Z-disc formation.

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.

C-terminally deleted fragments of 40-kDa earthworm actin modulator still show gelsolin activities

C‐ and N‐terminally truncated fragments of earthworm gelsolin were constructed, cloned and expressed in Escherichia coli. G‐actin‐binding properties of these fragments and their influences on the polymeric state of actin were investigated. A construct lacking a large part of the third segment [E(1–295)] supports actin nucleation similar to the complete protein and shows reduced actin fragmentation property, but is no longer Ca2+‐sensitive in its activity. The first and the second segments (E1 and E2) each contain one actin‐binding site. In contrast to human gelsolin, E1 in combination with a short N‐terminal region of E2 is not sufficient for the F‐actin‐severing activity of the protein.