Robust autophagy/mitophagy persists during mitosis - PubMed (original) (raw)

Robust autophagy/mitophagy persists during mitosis

Leyuan Liu et al. Cell Cycle. 2009.

Abstract

From microscopic observations of autophagosome content it has been argued that autophagy is shut down during mitosis to protect the relative short-lived organelles spindle and chromosomes from the process while they are contiguous with cytosol. However, without autophagy, buildup of dysfunctional mitochondria arising from the intense energy demands of mitosis potentially poses a hazard to accurate partition of chromosomes. Here we show using biochemical markers of autophagosomes and mitophagosomes and a blockade at the lysosomal clearance step that autophagy/mitophagy persists during mitosis at robust levels equal to interphase. This suggests a mechanism that insulates normal spindle and chromosomes from autophagy and potentially recognition of defects in spindle and chromosomes by the autophagic process.

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Figures

Figure 1

The autophagic pathway occurs in mitotic cells similar to interphase cells. (A) Punctate foci of GFP-LC3-containing autophagosomes at all mitotic substages revealed by overnight blockade of autophagosome clearance with lysosomal inhibitor NH4Cl (20 mM) or Bafilomycin A1 (BFA, 10 nM). The cell cycle stages for Hela cells stably expressing GFP-LC3 were identified by staining with DNA dye TO-PRO-3 iodide (1 μM) after cells were fixed and permealized as described. Total GFP-LC3 signal thus appears more intense in rounded mitotic cells. Scale bar, 10 μm in all panels. (B) Percentages of interphase or mitotic cells exhibiting GFP-LC3 punctate foci after treatment overnight with lysosomal inhibitors NH4Cl or Bafilomycin A1 (BFA). (C) Autophagosome accumulation revealed by LC3-II accumulation in mitotic cells. Wild type mitotic cells were enriched by synchronization with 2 mM thymidine for 20 hr followed by release from the thymidine blockade for 20 hr as described previously. NH4Cl was added to medium 12 hrs before cells were harvested in order to collect sufficient mitotic cells for analysis. Loosely attached mitotic cells (Shakeoff) were harvested by vigorous shake off from the remaining mixture (Attached) of interphase cells and mitotic cells attached to the dish. Equal amounts of protein from each sample were analyzed by immunoblot with the indicated antiserum. p-H3, phosphorylated histone H3 indicative of mitotic cells. (D) Specific accumulation of GFP-LC3-II and LC3-II in Hela cells stably expressing GFP-LC3 in response to NH4Cl or Bafilomycin A1 as in (A). Cell lysates were collected from treated cells without synchronization and fractionation. (E) Rapid accumulation of LC3-II in mitotic cells treated with NH4Cl. Mitotic cells were collected by shakeoff from wild type Hela cells that were released from thymidine blockade but further blocked in mitosis with 5 μM nocodazole for 20 hr as described previously and maintained in fresh medium containing the same concentration of nocodazole. The cells were treated with 20 mM of NH4Cl for the indicated times and used to prepared lysates for immunoblot. The intensity of β-actin served as the loading control. Ctrl, control.

Figure 2

Accumulation of mitophagosomes in mitotic cells in the presence of NH4Cl. (A) The colocalization of GFP-LC3 punctate foci (green) with mitochondria labeled with MitoTracker Red CMXRos dye (red) as previously described. Mitotic stages were identified with DAPI staining. The punctate foci of bright green GFP-LC3 in stably transfected Hela cells overlapping with weak red punctate foci of dysfunctional mitochondria was indicative of mitophagosomes. Colocalized punctate foci in white were revealed by analysis with an ImageJ ColocalizeRGB Plugin. Scale bar, 10 μm in all panels. (B) The colocalization of GFP-LC3 punctate foci with fragmented mitochondria in interphase cells stably expressing GFP-LC3 treated with lysosomal inhibitor NH4Cl and Bafilomycin A1. A small portion of a single interphase cell was enlarged and shown. White arrows point to individual mitochondria contained in autophagosomes (mitophagosomes). Scale bar: 5 μm. (C) Mitochondria accumulation as indicated by the citrate synthase activity. Mitotic cells collected and maintained as described in Figure 1E were treated with protein translation inhibitor cycloheximide (CHX, 1 μg/ml) and NH4Cl individually or in combination but for extended periods of time. The percentages of increase of citrate synthase activities at different times from the initial activity at time zero were plotted.

References

1. Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr. 2007;27:19–40. - PubMed
1. Mendez J, Zou-Yang XH, Kim SY, Hidaka M, Tansey WP, Stillman B. Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. Mol Cell. 2002;9:481–91. - PubMed
1. Sorger PK, Dobles M, Tournebize R, Hyman AA. Coupling cell division and cell death to microtubule dynamics. Curr Opin Cell Biol. 1997;9:807–14. - PubMed
1. Eskelinen EL, Prescott AR, Cooper J, Brachmann SM, Wang L, Tang X, et al. Inhibition of autophagy in mitotic animal cells. Traffic. 2002;3:878–93. - PubMed
1. Tasdemir E, Maiuri MC, Tajeddine N, Vitale I, Criollo A, Vicencio JM, et al. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy. Cell cycle. 2007;6:2263–7. - PubMed

Robust autophagy/mitophagy persists during mitosis - PubMed (original) (raw)