Androcam Is a Tissue-specific Light Chain for Myosin VI in the Drosophila Testis

Abstract

Myosin VI, a ubiquitously expressed unconventional myosin, has roles in a broad array of biological processes. Unusual for this motor family, myosin VI moves toward the minus (pointed) end of actin filaments. Myosin VI has two light chain binding sites that can both bind calmodulin (CaM). However unconventional myosins could use tissue-specific light chains to modify their activity. In the Drosophila testis, myosin VI is important for maintenance of moving actin structures, called actin cones, which mediate spermatid individualization. A CaM-related protein, Androcam (Acam), is abundantly expressed in the testis and like myosin VI, accumulates on these cones. We have investigated the possibility that Acam is a testis-specific light chain of Drosophila myosin VI. We find that Acam and myosin VI precisely colocalize at the leading edge of the actin cones and that myosin VI is necessary for this Acam localization. Further, myosin VI and Acam co-immunoprecipitate from the testis and interact in yeast two-hybrid assays. Finally Acam binds with high affinity to peptide versions of both myosin VI light chain binding sites. In contrast, although Drosophila CaM also shows high affinity interactions with these peptides, we cannot detect a CaM/myosin VI interaction in the testis. We conclude that Acam and not CaM acts as a myosin VI light chain in the Drosophila testis and hypothesize that it may alter the regulation of myosin VI in this tissue.The myosins constitute a superfamily of 18 classes (1 conventional and 17 unconventional) (1), all of which share sequence similarity in the motor domain. It is thought that they also share the ability to translocate along actin filaments. Myosins play roles in numerous cellular activities, such as division, endocytosis, cell movement, and organelle trafficking. In addition to the conserved motor domain, all myosins contain one or more conserved IQ motifs (consensus sequence IQXXXRGXXXR, where X is any amino acid) that bind calmodulin (CaM), 4 a ubiquitous calcium sensor protein, or CaM-like light chains (2-4). The ATPase activity and motility of unconventional myosins can be regulated by calcium binding to CaM or a CaMlike protein (5).Members of the myosin VI class appear to be unique among the unconventional myosins in their direction of translocation along actin. They move toward the pointed, or minus, end of actin filaments (6) whereas all other myosins tested move toward the barbed, or plus, end. Recent structural studies of pig myosin VI (7) indicate that this reversed directionality is associated with one of the two unique inserts in myosin VI, termed insert 2. This sequence is located between the converter region of the motor domain and the IQ motif, and its converter-proximal sequences are proposed to redirect the lever arm in the opposite direction relative to other myosins.Insert 2 is also proposed to perform a second, structural, role in myosin VI. In the pig crystal structure, its more C-terminal sequences form a continuous helix with the IQ motif, thus potentially exte...

Duplicated proteasome subunit genes in Drosophila and their roles in spermatogenesis

“…The generation of testis-specific genes by such a mechanism might indicate that some features of spermatogenesis require an increased amount of some particular gene product, or that there is a need for a version of the protein with an altered activity better adapted to its specific role in spermatogenic cells. The occurrence of testis-specific isoforms of various proteins generated by gene duplication events is not uncommon in Drosophila (for example, casein kinase 2 , mitochondrial translocase of the outer membrane (Hwa et al, 2004), TAFs , OXPHOS genes (Tripoli et al, 2005), myosin VI light chain (Frank et al, 2006), glycogen synthase kinase-3 (Kalamegham et al, 2007), eIF4G (Baker and Fuller, 2007;Franklin-Dumont et al, 2007), see also Mikhaylova et al, 2008)), and is also documented for some mammalian genes (for example, phosphoglycerate kinase (McCarrey and Thomas, 1987), cytochrome c (Hake et al, 1994), ornithine decarboxylase antizyme (Ivanov et al, 2000), TAF(II)250 (Wang and Page, 2002)). However, the degree to which the proteasome subunit genes have undergone such rapid proliferation in the Drosophila genome to produce testis-specific genes is remarkable.…”

Structure of androcam supports specialized interactions with myosin VI

“…1) reveals that the C-terminal lobe (C lobe) is more strongly conserved than the N-terminal lobe (N lobe), and although the androcam C lobe binds two Ca 2þ ions 40-fold more tightly than calmodulin, the N lobe binds weakly to only one Ca 2þ (11). Androcam interacts with both Insert 2 and IQ regions of Drosophila myosin VI in yeast two-hybrid analysis, but binds more weakly than calmodulin to an Insert 2 peptide (8). These prior studies demonstrate that the proteins are biochemically distinct but fail to illuminate how androcam might preferentially localize to the actin cones or might adjust the properties of myosin VI for its role in spermatogenesis.…”

“…In mammals, the light chain calmodulin binds to two myosin VI motifs within the lever arm: the myosin VI-specific Insert 2 sequence and an IQ motif (9,10). In the Drosophila ovary, calmodulin binds to myosin VI, but in the testis, androcam (and not calmodulin) coimmunoprecipitates with myosin VI (8). Androcam colocalizes with myosin VI at the flat leading edge of specialized structures (actin cones) as they move along the axonemes during spermatid individualization, and this localization is myosin VI-dependent (8).…”

“…In the Drosophila ovary, calmodulin binds to myosin VI, but in the testis, androcam (and not calmodulin) coimmunoprecipitates with myosin VI (8). Androcam colocalizes with myosin VI at the flat leading edge of specialized structures (actin cones) as they move along the axonemes during spermatid individualization, and this localization is myosin VI-dependent (8). Sequence alignment (Fig.…”

“…We are studying the structure and binding properties of the testis-specific calmodulin-like D. melanogaster protein androcam (6), which is conserved across the genus (7), to understand its role as a tissue-specific light chain for myosin VI in fly spermatogenesis (8). In mammals, the light chain calmodulin binds to two myosin VI motifs within the lever arm: the myosin VI-specific Insert 2 sequence and an IQ motif (9,10).…”