The Neuronal Calcium Sensor Protein Acrocalcin: A Potential Target of Calmodulin Regulation during Development in the Coral Acropora millepora Alejandro Reyes-Bermudez 1,2 , David J. Miller 1 , Susanne Sprungala 1 * 1 ARC Centre of Excellence for Coral Reef Studies and School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland, Australia, 2 Okinawa Institute of Science and Technology, Okinawa, Japan Abstract To understand the calcium-mediated signalling pathways underlying settlement and metamorphosis in the Scleractinian coral Acropora millepora, a predicted protein set derived from larval cDNAs was scanned for the presence of EF-hand domains (Pfam Id: PF00036). This approach led to the identification of a canonical calmodulin (AmCaM) protein and an uncharacterised member of the Neuronal Calcium Sensor (NCS) family of proteins known here as Acrocalcin (AmAC). While AmCaM transcripts were present throughout development, AmAC transcripts were not detected prior to gastrulation, after which relatively constant mRNA levels were detected until metamorphosis and settlement. The AmAC protein contains an internal CaM-binding site and was shown to interact in vitro with AmCaM. These results are consistent with the idea that AmAC is a target of AmCaM in vivo, suggesting that this interaction may regulate calcium-dependent processes during the development of Acropora millepora. Citation: Reyes-Bermudez A, Miller DJ, Sprungala S (2012) The Neuronal Calcium Sensor Protein Acrocalcin: A Potential Target of Calmodulin Regulation during Development in the Coral Acropora millepora. PLoS ONE 7(12): e51689. doi:10.1371/journal.pone.0051689 Editor: Eugene A. Permyakov, Russian Academy of Sciences, Institute for Biological Instrumentation, Russian Federation Received July 30, 2012; Accepted November 5, 2012; Published December 17, 2012 Copyright: ß 2012 Reyes-Bermudez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the School of Pharmacy and Molecular Sciences, James Cook University and the ARC Centre of Excellence for Coral Reef Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: susanne.sprungala@jcu.edu.au Introduction Scleractinian corals play important ecological roles, as they are responsible for the underlying framework of coral reefs, one of the most productive ecosystems on earth [1,2]. However, the molecular mechanisms underlying many aspects of their biology, including symbiosis, calcification and regeneration, are still poorly understood. Calcium metabolism and homeostasis are of partic- ular interest in corals in the context of calcification. Recent microarray studies [3,4,5,6] suggest that calcium-dependent signalling pathways may regulate metamorphosis, symbiosis and skeleton deposition in scleractinian corals. Consistent with this, clear counterparts of many of the molecules known to play key roles in calcium signalling and homeostasis in vertebrates are present in Acropora [7,8]. However, surprisingly little is known about either calcium metabolism or calcium-dependent signalling pathways in corals. Eukaryotes use changes in intracellular calcium concentration to regulate a diverse variety of cellular signalling pathways [9,10]. Calcium signalling is regulated by calcium itself via calcium- modulating proteins, which are involved in all aspects of cell function [11]. The EF-hand family is the most studied group of intracellular calcium-binding proteins able to implement the calcium signal or to buffer its cytosolic concentration [9,12]. Despite sequence and structural similarity, the responses of these ‘‘calcium sensors’’ to binding of calcium are diverse [13]. Upon calcium binding, this group of molecules typically undergoes topological changes within the EF-hand domain, a helix-loop-helix motif [14], enabling interaction with specific target proteins initiating a signalling cascade that will lead to specific cellular responses [13]. Calmodulin (CaM) is considered the most versatile ‘‘calcium sensor’’ due to its role regulating essential cellular processes such as cell cycle and calcium homeostasis across eukaryotes [15,16]. CaM sequences are known for several cnidarians [17,18,19,20] in- cluding Hydra magnipapillata, Nematostella vectensis and Acropora species [18] and CaM expression is up regulated during metamorphosis in the coral Montastraea faveolata [5]. Although CaM has been extensively investigated in the context of regulation of many calcium dependent processes, little is known about its interactions in early diverging metazoans and, as a key regulator of calcium- dependent processes, the identification of CaM targets may shed some light on the control of calcium carbonate deposition in corals as well as other processes such as metamorphosis and symbiont interactions. Furthermore, because Scleractinia represent an early diverging animal phylum, unravelling the roles of calcium- dependent process in corals may contribute to understanding the broader evolutionary history of calcium-dependent cellular path- ways. A number of transcripts encoding putative calcium sensor proteins were identified in the transcriptome of Acropora millepora [21], amongst which an uncharacterized NCS protein known here as Acrocalcin (AmAC) emerged as a putative AmCaM target as it contains a predicted CaM-binding site. In this study, we characterized AmCaM and AmAC expression profiles as well as the ability of the AmCaM and AmAC proteins to interact in vitro. PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e51689