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Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

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CMA is up-regulated in MA-deficient cells and stimulated by PDT-treatment. All controls represent incubation with hypericin without irradiation. (A) Confocal analysis of LAMP2A and Hsc70 immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) in 3T3 fibroblasts. DAPI was used for nuclear counterstaining. (B) Confocal analysis of Hsc70 (green, a and e) and LAMP2A (red b, f) co-immunostaining in untreated (a, b) and 6 hrs after PDT-treated (e, f) MEFs. The colocalization image is shown for the control (c) and the treated cells (g). DAPI was used as a nuclear counterstain and (d) and (h) present magnifications of the areas indicated in (c) and (g), respectively. The images are representative for approximately 70% of the cellular population after PDT. (C) Confocal analysis of LAMP2A immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) cells. MEF (a and b), Atg5–/– MEF (c and d). Red fluorescence represents LAMP2A immunostaining and blue fluorescence (DAPI) was used for nuclear counterstaining. The images are representative for approximately 70% of the cellular population after PDT. The graphs represent the distribution of LAMP2A+ vesicles respect to the nucleus. *P < 0.0001 for the Mann-Whitney test, ns: not significant. six cells were analysed per condition using the ImageJ software. The scale bar represents 15 μm for all the images shown.
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fig06: CMA is up-regulated in MA-deficient cells and stimulated by PDT-treatment. All controls represent incubation with hypericin without irradiation. (A) Confocal analysis of LAMP2A and Hsc70 immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) in 3T3 fibroblasts. DAPI was used for nuclear counterstaining. (B) Confocal analysis of Hsc70 (green, a and e) and LAMP2A (red b, f) co-immunostaining in untreated (a, b) and 6 hrs after PDT-treated (e, f) MEFs. The colocalization image is shown for the control (c) and the treated cells (g). DAPI was used as a nuclear counterstain and (d) and (h) present magnifications of the areas indicated in (c) and (g), respectively. The images are representative for approximately 70% of the cellular population after PDT. (C) Confocal analysis of LAMP2A immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) cells. MEF (a and b), Atg5–/– MEF (c and d). Red fluorescence represents LAMP2A immunostaining and blue fluorescence (DAPI) was used for nuclear counterstaining. The images are representative for approximately 70% of the cellular population after PDT. The graphs represent the distribution of LAMP2A+ vesicles respect to the nucleus. *P < 0.0001 for the Mann-Whitney test, ns: not significant. six cells were analysed per condition using the ImageJ software. The scale bar represents 15 μm for all the images shown.

Mentions: CMA has been shown to be involved in the selective removal of oxidized proteins in cells treated with H2O2 or the superoxide-generating drug paraquat [16]. Because activation of CMA after cellular stress is associated with a redistribution of CMA-active lysosomes from the cytosol towards the perinuclear region [16, 40], we evaluated the involvement of this lysosomal pathway by immunofluorescence microscopy. While untreated cells displayed a homogenous pattern of LAMP2A, the specific receptor for CMA, in the cytosol, PDT induced a marked redistribution of LAMP2A positive puncta to the perinuclear region of 3T3 cells (Fig. 6A) and MEFs (Fig. 6C). PDT induced also the relocalization of the CMA chaperone Hsc70 towards the perinuclear area (Fig. 6A), thus suggesting the recruitment of the CMA machinery in photosensitized cells. Moreover, a fraction of the total pool of LAMP2A positive lysosomes co-localized with Hsc70, thus indicating CMA activation by PDT in MA-competent cells (Fig. 6B).


Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

CMA is up-regulated in MA-deficient cells and stimulated by PDT-treatment. All controls represent incubation with hypericin without irradiation. (A) Confocal analysis of LAMP2A and Hsc70 immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) in 3T3 fibroblasts. DAPI was used for nuclear counterstaining. (B) Confocal analysis of Hsc70 (green, a and e) and LAMP2A (red b, f) co-immunostaining in untreated (a, b) and 6 hrs after PDT-treated (e, f) MEFs. The colocalization image is shown for the control (c) and the treated cells (g). DAPI was used as a nuclear counterstain and (d) and (h) present magnifications of the areas indicated in (c) and (g), respectively. The images are representative for approximately 70% of the cellular population after PDT. (C) Confocal analysis of LAMP2A immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) cells. MEF (a and b), Atg5–/– MEF (c and d). Red fluorescence represents LAMP2A immunostaining and blue fluorescence (DAPI) was used for nuclear counterstaining. The images are representative for approximately 70% of the cellular population after PDT. The graphs represent the distribution of LAMP2A+ vesicles respect to the nucleus. *P < 0.0001 for the Mann-Whitney test, ns: not significant. six cells were analysed per condition using the ImageJ software. The scale bar represents 15 μm for all the images shown.
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fig06: CMA is up-regulated in MA-deficient cells and stimulated by PDT-treatment. All controls represent incubation with hypericin without irradiation. (A) Confocal analysis of LAMP2A and Hsc70 immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) in 3T3 fibroblasts. DAPI was used for nuclear counterstaining. (B) Confocal analysis of Hsc70 (green, a and e) and LAMP2A (red b, f) co-immunostaining in untreated (a, b) and 6 hrs after PDT-treated (e, f) MEFs. The colocalization image is shown for the control (c) and the treated cells (g). DAPI was used as a nuclear counterstain and (d) and (h) present magnifications of the areas indicated in (c) and (g), respectively. The images are representative for approximately 70% of the cellular population after PDT. (C) Confocal analysis of LAMP2A immunostaining in untreated cells (a, c) or 6 hrs after PDT-treated (b, d) cells. MEF (a and b), Atg5–/– MEF (c and d). Red fluorescence represents LAMP2A immunostaining and blue fluorescence (DAPI) was used for nuclear counterstaining. The images are representative for approximately 70% of the cellular population after PDT. The graphs represent the distribution of LAMP2A+ vesicles respect to the nucleus. *P < 0.0001 for the Mann-Whitney test, ns: not significant. six cells were analysed per condition using the ImageJ software. The scale bar represents 15 μm for all the images shown.
Mentions: CMA has been shown to be involved in the selective removal of oxidized proteins in cells treated with H2O2 or the superoxide-generating drug paraquat [16]. Because activation of CMA after cellular stress is associated with a redistribution of CMA-active lysosomes from the cytosol towards the perinuclear region [16, 40], we evaluated the involvement of this lysosomal pathway by immunofluorescence microscopy. While untreated cells displayed a homogenous pattern of LAMP2A, the specific receptor for CMA, in the cytosol, PDT induced a marked redistribution of LAMP2A positive puncta to the perinuclear region of 3T3 cells (Fig. 6A) and MEFs (Fig. 6C). PDT induced also the relocalization of the CMA chaperone Hsc70 towards the perinuclear area (Fig. 6A), thus suggesting the recruitment of the CMA machinery in photosensitized cells. Moreover, a fraction of the total pool of LAMP2A positive lysosomes co-localized with Hsc70, thus indicating CMA activation by PDT in MA-competent cells (Fig. 6B).

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

Show MeSH
Related in: MedlinePlus