Skip to main content

Advertisement

SpringerLink
Log in

Distribution and Characterization of Rhogocyte Cell Types in the Mantle Tissue of Haliotis laevigata

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

Molluscan rhogocytes are known to be the only cells able to synthesize hemocyanin that is one of the largest respiratory proteins in nature. However, investigation of rhogocyte cells in vitro is limited due to difficulty in isolating and establishing marine cell culture. The aim of this study was to investigate the nature and distribution of rhogocyte cells of Haliotis laevigata in the mantle tissue with respect to the expression of the two known isoforms of hemocyanin. Rhogocyte cells were identified using immunofluorescence-fluorescence in situ hybridization (IF-FISH) that involved simultaneous staining of localized hemocyanin by a polyclonal antibody while the mRNA was hybridized with FISH probes. The distribution of rhogocyte cells was demonstrated using flow cytometry, followed by cell sorting with fluorescence-activated cell sorter (FACS) and confocal microscope imaging for further characterization. Our results suggested that the mantle tissue is dominated by two distinct populations of rhogocyte cells that synthesize hemocyanin type 1. Observation with confocal microscopy of both populations revealed hemocyanin localization in the periphery of the cell membrane. Cell population with higher antibody signal had irregular and elongated cell morphology with punctate mRNA probe signals. The second population with lower antibody signal had ovoid morphology and wide distribution of mRNA probe signals. We suggest that these populations represent two distinct phases of hemocyanin biosynthesis of a single isoform, which is closely related to Haliotis tuberculata type 1 hemocyanin (HtH1). The knowledge acquired in this study enhances the understanding of the biology of rhogocyte cells and biosynthesis of hemocyanin.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

FACS:

Fluorescence-activated cell sorting

IF:

Immunofluorescence

FISH:

Fluorescence in situ hybridization

mRNA:

Messenger ribonucleic acid

FITC:

Fluorescence isothiocyanate

HtH:

Haliotis tuberculata hemocyanin

HlH:

Haliotis laevigata hemocyanin

KLH:

Keyhole limpet hemocyanin

DAPI:

4′,6-Diamidino-2-phenylindole, dihydrochloride

References

  • Abuzakouk M, Feighery C, O’Farrelly C (1996) Collagenase and dispase enzymes disrupt lymphocyte surface molecules. J Immunol Methods 194:211–216

    Article  CAS  PubMed  Google Scholar 

  • Adachi K, Hirata T, Nishioka T, Sakaguchi M (2003) Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme. Comp Biochem Physiol Part B Biochem Mol Biol 134:135–141

  • Albrecht U, Keller H, Gebauer W, Markl J (2001) Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod Haliotis tuberculata. Cell Tissue Res 304:455–462

    Article  CAS  PubMed  Google Scholar 

  • Bani G, Delfino G (1974) Ultrastructure of the cells of the boundary layer of small vessels in the digestive gland of the gastropod pulmonate Eobania vermiculata (Müller). Monit Zool Ital - Ital J Zool 8:71–83

    Google Scholar 

  • Becker MI, Fuentes A, Del Campo M et al (2009) Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties. Int Immunopharmacol 9:330–339

    Article  CAS  PubMed  Google Scholar 

  • Boer HH, Sminia T (1976) Sieve structure of slit diaphragms of podocytes and pore cells of gastropod molluscs. Cell Tissue Res 170:221–229

    Article  CAS  PubMed  Google Scholar 

  • Chu C, Rana TM (2006) Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54. PLoS Biol 4:e210

    Article  PubMed Central  PubMed  Google Scholar 

  • Curtis JE, Hersh EM (1972) The human secondary immune response to keyhole limpet haemocyanin. Clin Exp Immunol 10:171–177

    PubMed Central  CAS  PubMed  Google Scholar 

  • Dallinger R, Chabicovsky M, Hödl E et al (2005) Copper in Helix pomatia (Gastropoda) is regulated by one single cell type: differently responsive metal pools in rhogocytes. Am J Physiol Regul Integr Comp Physiol 289:R1185–R1195

    Article  CAS  PubMed  Google Scholar 

  • Gebauer W, Harris JR, Geisthardt G, Markl J (1999) Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h. J Struct Biol 128:280–286

    Article  CAS  PubMed  Google Scholar 

  • Harper E (1980) Collagenases. Annu Rev Biochem 49:1063–1078

    Article  CAS  PubMed  Google Scholar 

  • Harris JR, Markl J (1999) Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 30:597–623

    Article  CAS  PubMed  Google Scholar 

  • Harris JR, Markl J (2000) Keyhole limpet hemocyanin: molecular structure of a potent marine immunoactivator. A review. Eur Urol 37(Suppl 3):24–33

    Article  CAS  PubMed  Google Scholar 

  • Haszprunar G (1996) The molluscan rhogocyte (pore-cell, blasenzelle, cellule nucale), and its significance for ideas on nephridial evolution. J Molluscan Stud 62:185–211

    Article  Google Scholar 

  • Herscowitz HB, Harold WW, Stavitsky AB (1972) Immunochemical and immunogenic properties of a purified keyhole limpet haemocyanin. Immunology 22:51–61

    PubMed Central  CAS  PubMed  Google Scholar 

  • Herscowitz HB, Diblasio RC, Rosenberg JB (1975) A hemolytic plaque assay for the detection of direct and indirect antibody-forming cells to keyhole limpet hemocyanin. J Immunol Methods 6:331–346

    Article  CAS  PubMed  Google Scholar 

  • Jones GW, Bowen ID (1979) The fine structural localization of acid phosphatase in pore cells of embryonic and newly hatched Deroceras reticulatum (Pulmonata: Stylommatophora). Cell Tissue Res 265:253–265

    Google Scholar 

  • Keller H, Lieb B, Altenhein B et al (1999) Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1). Organization of the =400 kDa subunit, and amino acid sequence of its functional units f, g and h. Eur J Biochem 264:27–38

    Article  CAS  PubMed  Google Scholar 

  • Lamm DL, DeHaven JI, Riggs DR et al (1993) Keyhole limpet hemocyanin immunotherapy of murine bladder cancer. Urol Res 21:33–37

    Article  CAS  PubMed  Google Scholar 

  • Lieb B, Dimitrova K, Kang H et al (2006) Red blood with blue-blood ancestry: intriguing structure of a snail hemoglobin. Proc Natl Acad Sci U S A 103:12011–12016

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Linn JF, Black P, Derksen K et al (2000) Keyhole limpet haemocyanin in experimental bladder cancer: literature review and own results. Eur Urol 37(Suppl 3):34–40

    Article  CAS  PubMed  Google Scholar 

  • Manubens A, Salazar F, Haussmann D et al (2010) Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism. Cell Tissue Res 342:423–435

    Article  CAS  PubMed  Google Scholar 

  • Marigómez I, Soto M, Cajaraville MP et al (2002) Cellular and subcellular distribution of metals in molluscs. Microsc Res Tech 56:358–392

    Article  PubMed  Google Scholar 

  • Maroney PA, Yu Y, Nilsen TW (2006) MicroRNAs, mRNAs, and translation. Cold Spring Harb Symp Quant Biol 71:531–535

    Article  CAS  PubMed  Google Scholar 

  • Martin GG, Bessette T, Martin A et al (2010) Morphology of epithelial cells lining the digestive tract of the giant keyhole limpet, Megathura crenulata (Mollusca; Vetigastropoda). J Morphol 271:1134–1151

    Article  PubMed  Google Scholar 

  • Martin AM, Martin GG, Butler R, Goffredi SK (2011) Synthesis of keyhole limpet hemocyanin by the rhogocytes of Megathura crenulata. Invertebr Biol 130:302–312

    Article  Google Scholar 

  • Masson-Pévet M, Jongsma HJ, De Bruijne J (1976) Collagenase- and trypsin-dissociated heart cells: a comparative ultrastructural study. J Mol Cell Cardiol 8:747–757

    Article  PubMed  Google Scholar 

  • Moltedo B, Faunes F, Haussmann D et al (2006) Immunotherapeutic effect of Concholepas hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among hemocyanins. J Urol 176:2690–2695

    Article  CAS  PubMed  Google Scholar 

  • Musselli C, Livingston PO, Ragupathi G (2001) Keyhole limpet hemocyanin conjugate vaccines against cancer: the Memorial Sloan Kettering experience. J Cancer Res Clin Oncol 127(Suppl 2):R20–R26

    Article  CAS  PubMed  Google Scholar 

  • Skelding JM, Newell PF (1975) On the functions of the pore cells in the connective tissue of terrestrial pulmonate molluscs. Cell Tissue Res 156:381–390

    Article  CAS  PubMed  Google Scholar 

  • Sminia T (1972) Structure and function of blood and connective tissue cells of the fresh water pulmonate Lymnaea stagnalis studied by electron microscopy and enzyme histochemistry. Z Zellforsch Mikrosk Anat 130:497–526

    Article  CAS  PubMed  Google Scholar 

  • Sminia T, Boer HH (1973) Haemocyanin production in pore cells of the freshwater snail Lymnaea stagnalis. Z Zellforsch Mikrosk Anat 145:443–445

    Article  CAS  PubMed  Google Scholar 

  • Steinberg MS, Armstrong PB, Granger RE (1973) On the recovery of adhesiveness by trypsin-dissociated cells. J Membr Biol 13:97–128

    Article  CAS  PubMed  Google Scholar 

  • Streit K, Jackson D, Degnan BM, Lieb B (2005) Developmental expression of two Haliotis asinina hemocyanin isoforms. Differentiation 73:341–349

    Article  CAS  PubMed  Google Scholar 

  • Swerdlow RD, Ebert RF, Lee P et al (1996) Keyhole limpet hemocyanin: structural and functional characterization of two different subunits and multimers. Comp Biochem Physiol B Biochem Mol Biol 113:537–548

    Article  CAS  PubMed  Google Scholar 

  • Van Holde KE, Miller KI (1982) Haemocyanins. Q Rev Biophys 15:1–129

    Article  PubMed  Google Scholar 

  • Van Hoof D, Lomas W, Hanley MB, Park E (2014) Simultaneous flow cytometric analysis of IFN-γ and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation. Cytometry A. doi:10.1002/cyto.a.22521

    PubMed  Google Scholar 

  • Walter RJ (1986) Clostridial collagenase. A chemoattractant for human neutrophils. Inflammation 10:347–361

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was funded by the Australian Research Council (grant no. LP100100799) and Marine Biotechnology Australia Pty Ltd. We are also thankful to the Malaysian government for the scholarship award to Fareed Sairi, the Flinders University Antibody Facility for antibody production, Centenary Institute for providing access to their flow cytometry facility, and the Australian Centre for Microscopy and Microanalysis (ACMM) for the confocal microscopy imaging.

Author information

Authors and Affiliations

  1. School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, 2006, Australia

    Fareed Sairi, Peter Valtchev, Vincent G. Gomes & Fariba Dehghani

  2. School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Malaysia

    Fareed Sairi

Authors
  1. Fareed Sairi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Valtchev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vincent G. Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fariba Dehghani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fariba Dehghani.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sairi, F., Valtchev, P., Gomes, V.G. et al. Distribution and Characterization of Rhogocyte Cell Types in the Mantle Tissue of Haliotis laevigata . Mar Biotechnol 17, 168–179 (2015). https://doi.org/10.1007/s10126-014-9605-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-014-9605-9

Keywords

Search

Navigation