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Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis

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Abstract

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.

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Abbreviations

Apaf-1:

apoptosis protease-activating factor-1

Bcl-2:

B cell leukemia/lymphoma-2

Caspases:

cysteinyl-directed aspartate-specific proteases

DAPI:

4′,6-diamidino-2-phenylindole·2HCl

Hsf-1:

heat-shock factor 1

Hsps:

heat shock proteins

RBP:

rat brain proteins

RCH:

rapid cold-hardening

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TUNEL:

terminal deoxynucleotidyl transferase (TdT)-mediated uridine 5′-triphosphate- biotin (dUTP) nick end labeling

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Acknowledgments

This research was supported by NSF grant #IOB-0416720. We thank Katia Del Rio-Tsonis, Natalia Vergara and Juanita Constible for a critical reading of the manuscript.

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  1. Department of Zoology, Miami University, Oxford, OH, 45056, USA

    Shu-Xia Yi, Clifford W. Moore & Richard E. Lee Jr.

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  1. Shu-Xia Yi
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Yi, SX., Moore, C.W. & Lee, R.E. Rapid cold-hardening protects Drosophila melanogaster from cold-induced apoptosis. Apoptosis 12, 1183–1193 (2007). https://doi.org/10.1007/s10495-006-0048-2

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