Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata) Carlos A. Dı´az-Balzac1,2, Mar´ıa I. La´ zaro-Pen˜ a1,2, Enrique M. Garcı´a-Rivera2, Carlos I. Gonza´ lez2, Jose´ E. Garcı´a-Arrara´ s2* 1 Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Biology, University of Puerto Rico-R´ıo Piedras Campus, R´ıo Piedras, San Juan, Puerto Rico Abstract Members of the calbindin subfamily serve as markers of subpopulations of neurons within the vertebrate nervous system. Although markers of these proteins are widely available and used, their application to invertebrate nervous systems has been very limited. In this study we investigated the presence and distribution of members of the calbindin subfamily in the sea cucumber Holothuria glaberrima (Selenka, 1867). Immunohistological experiments with antibodies made against rat calbindin 1, parvalbumin, and calbindin 2, showed that these antibodies labeled cells and fibers within the nervous system of H. glaberrima. Most of the cells and fibers were co-labeled with the neural-specific marker RN1, showing their neural specificity. These were distributed throughout all of the nervous structures, including the connective tissue plexi of the body wall and podia. Bioinformatics analyses of the possible antigen recognized by these markers showed that a calbindin 2-like protein present in the sea urchin Strongylocentrotus purpuratus, corresponded to the calbindin-D32k previously identified in other invertebrates. Western blots with anti-calbindin 1 and anti-parvalbumin showed that these markers recognized an antigen of approximately 32 kDa in homogenates of radial nerve cords of H. glaberrima and Lytechinus variegatus. Furthermore, immunoreactivity with anti-calbindin 1 and anti-parvalbumin was obtained to a fragment of calbindin-D32k of H. glaberrima. Our findings suggest that calbindin-D32k is present in invertebrates and its sequence is more similar to the vertebrate calbindin 2 than to calbindin 1. Thus, characterization of calbindin-D32k in echinoderms provides an important view of the evolution of this protein family and represents a valuable marker to study the nervous system of invertebrates. Citation: D´ıaz-Balzac CA, La´zaro-Pen˜ a MI, Garc´ıa-Rivera EM, Gonza´lez CI, Garc´ıa-Arrara´s JE (2012) Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata). PLoS ONE 7(3): e32689. doi:10.1371/journal.pone.0032689 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received November 23, 2011; Accepted January 29, 2012; Published March 7, 2012 Copyright: 2012 D´ıaz-Balzac 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: This work was supported by the National Science Foundation (IOS-0842870) and the National Institutes of Health (NIH, 1SC1GM084770-01 and 1R03NS065275-01). CADB and EMGR were funded by the University of Puerto Rico Rio Piedras Campus (UPR-RP) Minority Access to Research Career (MARC) Program (5T34GM007821) and MILP by the UPR-RP Research Initiative for Scientific Enhancement (RISE) Program (2R25GM061151). We also acknowledge partial support from NIH Research Centers in Minority Institutions (NIH-RCMI, RRO-3641-01) and the University of Puerto Rico. 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: jegarcia@hpcf.upr.edu Introduction EF-hand domain-containing calcium-binding proteins (CBP) of the calbindin and parvalbumin subfamilies have been used during the last three decades as markers of subpopulation of neurons in the nervous system of vertebrates [1–11]. Despite the vast interest in these proteins, their study in non-vertebrate species has not been widely pursued. The first major reports in non-vertebrates were conducted in Loligo pealeii (Lesueur, 1821) (Teuthida, Cephalopoda) [12] and Drosophila melanogaster (Meigen, 1830) (Diptera, Insecta) [13]. In the former a protein biochemically similar to calbindin 1 was characterized, though its sequence is not yet available. In the latter, the calbindin-D32k gene of Drosophila was cloned. This protein presents 42% and 37% homology to chicken calbindin 2 and calbindin 1, respectively [13]. Since then, sequences similar to calbindin-D32k have been identified in many representatives of Insecta as part of the whole genome sequencing being performed to study their evolution. So far, no other CBP of the calbindin or parvalbumin subfamilies has been reported to be present in a non-vertebrate organism. The calbindin subfamily of EF-hand domain-containing CBP is characterized by having six EF-hand motifs and is made up by calbindin 1 (calbindin-D28k), calbindin 2 (calretinin), and secretagogin. These proteins function as calcium buffers, but their physiological role remains unknown [14,15]. In vertebrates, specifically in mammals, calbindin 1, parvalbumin and calbindin 2 are present in different populations of neurons and interneurons of mice adult spinal cord [10]. In amphibians, differential immunoreactivity to anti-calbindin 1 and anti-calbindin 2 in cells and fibers throughout the brainstem serves to determine the localization and signature of many cell groups [9]. Lastly, calbindin 1 and calbindin 2-like immunoreactivities are present in the retina of the lamprey, the basal vertebrate studied at present [16]. In invertebrates, only two members of the calbindin subfamily have been identified, calbindin-D32k [13] and secreta- gogin [17]. In Drosophila, calbindin-D32k is selectively distributed PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32689