Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster

Abstract

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners.

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

“…Class V myosins from budding yeast, fission yeast, Drosophila and chicken brains associate with the ELC (D'Souza et al, 2001;Espindola et al, 2000;Franke et al, 2006;Luo et al, 2004;Stevens and Davis, 1998) as do Drosophila myosin-VI and -VIIA associate with the ELC (Franke et al, 2006), whereas human myosin-X and Toxoplasma gondii myosin-XIV employ novel light chains (Herm-Gotz et al, 2002;Rogers and Strehler, 2001). …”

N-Methyl-D-aspartate Receptor Subunits Are Non-myosin Targets of Myosin Regulatory Light Chain

“…This finding has attracted much attention because ELC in organisms such as S. cerevisiae, Schizosaccharomyces pombe, and Drosophila melanogaster have several binding partners (11). These include class II myosin heavy chains (Myo1p in S. cerevisiae (12,13), Myp2 and Myo2 in S. pombe (14), and zipper in D. melanogaster (11)), myosin V heavy chains (11,15), additional unconventional myosins (VI and VIIa in D. melanogaster (11)), cytoskeletal IQ containing GTPase-activating protein-like proteins (16 -18), and a microtubule-associated protein (11).…”

“…This viewpoint has been facilitated by clear differences in the spatial and temporal distribution and/or stability of the ELC versus RLC and myosin II heavy chain in organisms such as Dictyostelium (57), the yeasts S. cerevisiae and S. pombe (13,16,17), and D. melanogaster (11). These kinds of studies are more difficult in the mammalian central nervous system as expression of components of the myosin II complex is under the control of more than one gene, thus making it difficult to track each polypeptide.…”

“…Differential staining patterns observed with RLC and non-muscle myosin II-B heavy chain antibodies in brain also indicate that heterogeneity in the composition of native myosin II complexes is likely, despite comparable expression of RLC, ELC, and NMHC-IIB in mouse cortex, hippocampus, and cerebellum (52). In D. melanogaster, which has a single gene encoding the ELC (essential light chain-cytoplasmic), RLC (spaghetti squash), and heavy chain (zipper), at least four alternate binding partners for the ELC have been identified (11). However, all three components of the myosin II complex appear to be expressed in relatively constant stoichiometric amounts, and by far the majority of both light chains is bound to myosin II heavy chain; using an immunodepletion strategy greater than 90% of ELC and greater than 95% of RLC were bound to myosin II heavy chain in wild-type embryos (11).…”

“…In D. melanogaster, which has a single gene encoding the ELC (essential light chain-cytoplasmic), RLC (spaghetti squash), and heavy chain (zipper), at least four alternate binding partners for the ELC have been identified (11). However, all three components of the myosin II complex appear to be expressed in relatively constant stoichiometric amounts, and by far the majority of both light chains is bound to myosin II heavy chain; using an immunodepletion strategy greater than 90% of ELC and greater than 95% of RLC were bound to myosin II heavy chain in wild-type embryos (11). These authors conclude that although the majority of both light chains are associated with non-muscle myosin II heavy chain, pools of free light chain and/or light chain bound to other proteins are present (11).…”