Myomesin belongs to the family of so-called myosin-binding proteins whose distinguishing property is their interaction with the thick filament protein myosin (Weber et al., 1993
). Although myosin filaments stretch across the entire A-band in a staggered manner, the myosin-binding proteins have restricted localization patterns. Analysis by electron microscopy has shown that myomesin is located exclusively in the central area of the M-band (Obermann et al., 1996
), whereas MyBP-C, for example, is localized in several consecutive stripes in the A-band (Craig and Offer, 1976
). Therefore, it is likely that apart from the basic myosin-binding activity, interactions with other sarcomeric components may be needed for the precise targeting of these proteins.
Our results show that in cultured cardiomyocytes, two domains in the N-terminal part of rat myomesin are targeted to specific sites in the sarcomere. The unique head domain of rat myomesin localized to a central area within the A-band, and the adjacent immunoglobulin-like domain localized to the M-band. The other parts of myomesin did not interact with the sarcomere and were diffusely distributed in the cytoplasm. So far, no binding partner of the C-terminal immunoglobulin-like domains of myomesin has been identified, but the fibronectin type III domains 4–6 were shown to bind titin in a solid-phase overlay assay (Obermann et al., 1997
). Therefore, it is surprising that the construct cMy4–8 did not localize to the M-band in cardiomyocytes. A suggested function of titin is that of a sarcomeric ruler with which other proteins can interact to find their proper place in the sarcomere (Trinick, 1994
). The interaction of myomesin with titin would be well suited to localize the protein to the M-band, but our epitope-tagging experiments show that this is not the case. It is possible that in the mature sarcomere not all binding sites are accessible for exogenously expressed fragments or that the interaction is regulated by additional factors such as phosphorylation. For example, phosphorylation of the exogenous fragment cMy4–8 may prevent its interaction with titin and therefore its incorporation into the M-band, because it has been shown that the in vitro interaction of myomesin with titin is abolished by phosphorylation of a linker connecting two of the fibronectin type III domains (Obermann et al., 1997
In both species investigated, the second domain of myomesin was targeted to the M-band. Furthermore, it was absolutely essential for M-band targeting because only fragments containing this domain localized to the M-band; however, it was observed that the fragment rMy2 localized in slightly broader stripes than the fragment cMy2, whereas the fragment rMy2–3 clearly localized to the M-band. The somewhat enhanced targeting of the two-domain fragment may simply be due to a stabilization of the interacting domain rMy2 by its neighboring domain, especially in view of the fact that the corresponding chicken myomesin fragment cMy2 was clearly targeted to the M-band. On the other hand, the enhanced targeting may result from a cooperative interaction of both immunoglobulin-like domains. The need for cooperative interactions to ensure targeting to sarcomeric sites has been observed before. In the case of MyBP-C, the C-terminal immunoglobulin-like domain bound to myosin in biochemical assays (Okagaki et al., 1993
), but only a larger fragment encoding the three C-terminal domains was correctly incorporated into the A-bands of primary chicken myotubes (Gilbert et al., 1996
). Interestingly, this three-domain fragment also bound to titin (Freiburg and Gautel, 1996
), which suggests that MyBP-C needs both interactions for its incorporation into the A-band. In M-protein, the cooperative action of two immunoglobulin-like domains is needed for binding to myosin (Obermann et al., 1998
In addition, the observation that in the solid-phase overlay assay the complete N-terminal fragment had a stronger affinity for myosin than the unique head domain alone suggests that the second domain may bind cooperatively with the head domain to myosin. Thus, it may be needed to restrict myomesin to the M-band. The binding site of myomesin was mapped to the C-terminal portion of the myosin rod (Obermann et al., 1997
). The rod portions of myosin filaments from two adjacent A-bands overlap in an antiparallel manner in the M-band region (Huxley, 1963
). No myosin heads are present in this region of overlap, which is also called the “bare zone.” The unique configuration of antiparallel, overlapping myosin rods may lead to binding sites that are present only in the bare zone and not in the cross-bridge zone of the thick filament. The binding of the unique head domain to this region could explain the wider localization pattern of the constructs rMy1 and rMy1 + 3. Studies using electron microscopy will be needed to prove this hypothesis.
The unique head domain of chicken myomesin did not localize to the bare zone but instead was diffusely distributed in the cytoplasm. Although this may be a result of misfolding or degradation, the observation that cMy1 retained its ability to bind myosin when expressed in vitro suggests that the domain is capable of folding. Therefore, the difference in targeting behavior of the unique head domains in cultured cardiomyocytes may be due to a species-specific difference that cannot be explained by the biochemical binding assays. A comparison of the head domain sequences of different species reveals strong differences, such as a repeat motif composed of the residues KQSTAS that is present in varying copy numbers in the head domains of human, mouse, and rat myomesin but is absent from the head domain of chicken myomesin (Bantle et al., 1996
). The repeat motif may be involved in myosin binding, and its absence in chicken myomesin may be responsible for the lack of targeting observed with cMy1. Possibly, such species-specific differences in sarcomeric proteins may lead to different ultrastructures of the M-band (Carlsson and Thornell, 1987
; Pask et al., 1994
None of the constructs containing the second domain were incorporated into the NSMFs present in cardiomyocytes. NSMFs are filamentous extensions of myofibrils that contain some nonsarcomeric and sarcomeric proteins, such as titin and sarcomeric α-actinin, but no myomesin or MyBP-C (Schultheiss et al., 1990
). Therefore, NSMFs probably do not yet have all the binding sites that are present in mature sarcomeres. The fact that the second domain did not localize to NSMFs may indicate either that they do not contain the binding partner or alternatively that the binding partner is not yet in the configuration required to bind myomesin. For example, in mature myofibrils, titin filaments overlap in an antiparallel manner in the M-band (Obermann et al., 1996
). This overlap may lead to unique binding sites that are needed for the interaction with M-band proteins and may not be present in precursor structures such as the NSMFs. Several domains of human myomesin have been mapped for binding to titin using solid-phase overlay assays, and no affinity for titin was found in the case of the second domain (Obermann et al., 1997
); however, because the second domain does not bind to titin present in NSMFs either, a complex of overlapping titin molecules and/or other M-band proteins may be needed for an interaction with myomesin. This hypothesis is supported by studies performed in differentiating human skeletal muscle cells in vitro where evidence for a rearrangement of the C-terminal portion of titin on integration into the M-band of mature sarcomeres has been found (van der Ven and Fürst, 1997
Clearly, the interactions between myomesin and other sarcomeric proteins that take place during the assembly of myofibrils cannot be explained by simply studying their binding behavior in biochemical assays; therefore, assays that investigate the interactions in the cellular environment are indispensable. These studies may lead to the identification of a yet unknown protein that is necessary for restriction of myomesin to the M-band. The question of whether myomesin is essential as a linker between the filament systems and whether it has additional properties can be answered only by combining both approaches. It is hoped that further characterization of the second domain and its binding partners will lead to a better understanding of the role of myomesin in the assembly and maintenance of myofibrils.