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New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach

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Abstract

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity.

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Acknowledgements

The authors thank Dr. Dwight Lynn (INSell Consulting, 247 Lynch Rd, Newcastle, ME 04553, USA) for providing the IPLB-LdFB cell line.

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Authors and Affiliations

  1. Department of Animal Biology, University of Modena and Reggio Emilia, Via Campi 213/D, 41100, Modena, Italy

    Davide Malagoli & Enzo Ottaviani

  2. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

    Federica Boraldi, Giulia Annovi & Daniela Quaglino

Authors
  1. Davide Malagoli
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  2. Federica Boraldi
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  3. Giulia Annovi
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  4. Daniela Quaglino
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  5. Enzo Ottaviani
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Corresponding author

Correspondence to Davide Malagoli.

Additional information

This work was supported by an F.A.R. grant from the University of Modena and Reggio Emilia (D.M. and E.O.) and by an “Experimental approaches to the study of evolution” grant from the Department of Animal Biology of the University of Modena and Reggio Emilia (D.M.).

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Fig7

Supplementary Fig. S1 Schematic representation of the experimental design followed to obtain the conditioned media (C-CM and oligo A-CM) and their successive utilization for various experimental procedures (2D-GE two-dimensional gel electrophoresis, C-CM conditioned medium, oligo A-CM medium conditioned for 24 h by cells exposed previously to 10 μM oligomycin A for 2 h) (GIF 64 KB)

High resolution image (TIF 269 KB)

Fig8

Supplementary Fig. S2 Evaluation of cell viability in control and oligomycin A-treated IPLB-LdFB fat body cells at the beginning (time 0) and at the end (24 h) of the conditioning phase. *P<0.05 vs control (GIF 16 KB)

High resolution image (TIF 54.6 KB)

Fig9

Supplementary Fig. S3 Effects of oligo A-CM on untreated IPLB-LdFB cells. At the beginning of the experiment (a), cells present their typical round shape and are large (approximately 20 μm in diameter). Reduction of the cytoplasm is visible after 24 h (b) in the conditioned medium and is clearly observable also after 48 h (c) and 72 h (d) of incubation. During incubation in oligo A-CM, IPLB-LdFB cells gradually lose their flat shape and become more spherical. IPLB-LdFB cells incubated for 24 h (e) and 72 h (f) with C-CM show an increasing number of cytoplasmic blebs that cannot be related either to apoptotic figures or to the treatment since they represent the typical morphology of IPLB-LdFB cells cytocentrifuged 72 h after the last passage in medium, independent of the use of unconditioned medium, C-CM or oligo A-CM. Bar 20 μm (GIF 496 KB)

High resolution image (TIF 12 MB)

Fig10

Supplementary Fig. S4 Light microscopy of semithin sections from IPLB-LdFB cells exposed to C-CM for 24 h (a) or 72 h (c) and to oligo A-CM for 24 h (b) or 72 h (d). Note the progressive shrinkage of the cell surface in the presence of oligo A-CM, as clearly demonstrated by the morphometric analysis (f). Cells grown for 24 h in the presence of 10 nM oligomycin A (e) did not show any of the significant morphological alterations observable in b. Data in f are expressed as mean values ± SE. Bar 30 μm. **P<0.01, ***P<0.001 for oligo A-CM vs C-CM at the same exposure time (GIF 544 KB)

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Malagoli, D., Boraldi, F., Annovi, G. et al. New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach. Cell Tissue Res 336, 107–118 (2009). https://doi.org/10.1007/s00441-008-0748-8

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