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Apoptosis

, Volume 12, Issue 7, pp 1183–1193 | Cite as

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

  • Shu-Xia Yi
  • Clifford W. Moore
  • Richard E. LeeJr.
Article

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.

Keywords

Drosophila melanogaster Apoptosis TUNEL assay Rapid cold-hardening Cold shock 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Shu-Xia Yi
    • 1
  • Clifford W. Moore
    • 1
  • Richard E. LeeJr.
    • 1
  1. 1.Department of ZoologyMiami UniversityOxfordUSA

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