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beta-Thymosins and Hemocyte Homeostasis in a Crustacean
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
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2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 4, e60974- p.Article in journal (Refereed) Published
Abstract [en]

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (T beta 4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this beta-subunit of ATP synthase. Here, we identified five different beta-thymosins from Pacifastacus leniusculus, designated Pl-beta-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-beta-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the b-subunit of ATP-synthase, and Pl-beta-thymosin1, but not Pl-beta-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-beta-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-beta-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-beta-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-beta-thymosin2 had a similar but transient effect. Both Pl-beta-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-beta-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-beta-thymosin1 and Pl-beta-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.

Place, publisher, year, edition, pages
2013. Vol. 8, no 4, e60974- p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-200355DOI: 10.1371/journal.pone.0060974ISI: 000317717300166OAI: diva2:623577
Available from: 2013-05-28 Created: 2013-05-27 Last updated: 2013-11-06Bibliographically approved
In thesis
1. Melanization and Hemocyte Homeostasis  in the Freshwater Crayfish, Pacifastacus leniusculus
Open this publication in new window or tab >>Melanization and Hemocyte Homeostasis  in the Freshwater Crayfish, Pacifastacus leniusculus
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions.

In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1086
melanization, prophenoloxidase, caspase, hematopoiesis, thymosin, astakine, circadian rhythm, reactive oxygen species, tyrosinase, prokineticin
National Category
urn:nbn:se:uu:diva-209209 (URN)978-91-554-8774-4 (ISBN)
Public defence
2013-11-28, Lindahlssalen, EBC, Norbyvägen 18A, Uppsala, 10:00 (English)
Available from: 2013-11-06 Created: 2013-10-15 Last updated: 2016-05-09

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