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

Deborah J Frank,Stephen R Martin,Bridget N.T Gruender,Yung-Sheng R Lee,Rebecca A Simonette,Peter M Bayley,Kathryn G Miller,Kathleen M Beckingham

doi:10.1074/jbc.m602094200

Deborah J Frank, Stephen R Martin + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m602094200

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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.

Highlights

Where X is any amino acid) that bind calmodulin (CaM),4 a ubiquitous calcium sensor protein, or CaM-like light chains [2,3,4]
In Vivo Interactions of Acam with Myosin VI—During Drosophila spermatogenesis, myosin VI localizes to the fronts of actin cones, structures that are crucial to the individualization step in forming mature sperm [18]
We have presented four lines of evidence supporting the hypothesis that Acam serves as a light chain for myosin VI in peptide: protein complexes prevented the determination of a the Drosophila testis

Summary

The abbreviations used are

CaM, calmodulin; X-gal, 5-bromo-4-chloro3-indolyl-␤-D-galactopyranoside; Acam, Androcam; GST, glutathione S-transferase; C-Acam, C-terminal-half of Acam. Androcam Is a Myosin VI Light Chain mammalian cells [11, 12], protein localization and stabilization in neuroblasts and migrating ovarian cells in Drosophila melanogaster [13, 14], maintenance of stereocilia in the mammalian inner ear [15, 16], and creation or preservation of an actin barrier during spermatogenesis in the nematode Caenorhabditis elegans [17] It is required for the stability and continued movement of an actin structure used during spermatid individualization in Drosophila [18, 19]. In vitro experiments demonstrate strong binding of CaM to the relevant myosin VI peptides, we cannot detect an interaction between these two proteins in the testis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION