doi: 10.1038/ncomms8439.
A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure
Affiliations
- PMID:26074490
- PMCID: PMC4490561
- DOI: 10.1038/ncomms8439
Item in Clipboard
A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure
Georgia K Atkin-Smith et al. Nat Commun..
Display options
Format
Display options
Format
Abstract
Disassembly of apoptotic cells into smaller fragments (a form of extracellular vesicle called apoptotic bodies) can facilitate removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear. While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a 'beads-on-a-string' structure. Strikingly, the 'beads' are frequently sheared off the 'string' to form apoptotic bodies. Generation of apoptotic bodies via this mechanism can facilitate a sorting process and results in the exclusion of nuclear contents from apoptotic bodies. Mechanistically, generation of 'beads-on-a-string' protrusion is controlled by the level of actomyosin contraction and apoptopodia formation. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (an antidepressant) to block the formation of 'beads-on-a-string' protrusions and apoptotic bodies. These data uncover a new mechanism of apoptotic body formation in monocytes and also compounds that can modulate this process.
Figures
(a) Time-lapse images monitoring THP-1 monocytes undergoing ultraviolet-induced apoptosis. (b) Quantitation of live microscopy data to determine the percentage of apoptotic THP-1 cells that form membrane bleb and/or beaded apoptopodia during 4 h of time-lapse imaging (n=3). (c) Upper, isolation of primary human CD14+ cells from peripheral blood mononuclear cells. Lower, primary CD14+ cells undergoing spontaneous apoptosis during serum starvation. DIC images of apoptotic THP-1 cells forming uniform beaded apoptopodia (d) or non-uniform beaded apoptopodia (e) 2 h post ultraviolet irradiation. Quantitation of the diameter of vesicle-like structures on each type of beaded apoptopodia is shown on the right. (f) Fragmentation of beaded apoptopodia from apoptotic THP-1 and primary CD14+ cells under cultured conditions. (g) Images of apoptotic bodies stained with annexin V (green). (h) Flow cytometry analysis showing each type of cells and apoptotic particles gated according to Supplementary Fig. 3. (i) TO-PRO-3 dye uptake in viable and annexin V+ apoptotic THP-1 cells (n=3). (j) TO-PRO-3 uptake and formation of apoptotic bodies from apoptotic THP-1 cells treated with the PANX1 inhibitor trovafloxacin (n=3). (k) Quantitation of the percentage of untreated or trovafloxacin-treated apoptotic THP-1 cells that forms beaded apoptopodia (n=3). Error bars represent s.e.m. Data are representative of at least two independent experiments.P=0.07, unpaired Student's two-tailedt-test.
Localization of surface CD14 (a) and non-targeted intracellular GFP (b) in viable and apoptotic THP-1 monocytic cells. (c) Localization of Hoechst 33342, LysoTracker Red and MitoTracker Green staining in viable and apoptotic THP-1 cells. (d) Venn diagram of differentially abundant proteins in whole apoptotic sample and apoptotic body-enriched sample (a list of proteins identified in the proteomic analysis is shown in Supplementary Data 1). A total of 562 proteins were of low abundance in apoptotic body-enriched sample compared with whole apoptotic sample. Magenta and turquoise arrows indicate proteins that are in low or high abundance in apoptotic body-enriched sample compared with whole apoptotic sample, respectively. (e) Subcellular components depleted in apoptotic body-enriched sample compared with whole apoptotic sample are depicted based on FunRich analysis software.(f) Levels of histone H3, HMGB1 and β-actin in whole apoptotic sample and apoptotic body-enriched sample. (a–c,f) Data are representative of at least three independent experiments.
(a) Schematic of Jurkat cells (T cell line) undergoing apoptosis under conditions when actomyosin contraction and/or PANX1 functions are impaired based on time-lapse microscopy. (b) Images of cells at late stage of apoptosis when actomyosin contraction and/or PANX1 functions are blocked by pharmacological compounds. (c) Uptake of TO-PRO-3 by apoptotic cells treated with cytochalasin D (Cyto-D) and/or trovafloxacin. (d) Time-lapse images monitoring progression of apoptotic cell morphology when actomyosin contraction and PANX1 functions are blocked by pharmacological compounds.(e) Formation of beaded apoptopodia from non-beaded-apoptopodia. (f) Formation of smaller apoptotic bodies through fragmentation of beaded apoptopodia (n=3). Error bars represent s.e.m. Data are representative of at least two independent experiments.
(a) Schematic representation of the drug screen approach to identify compounds that could inhibit the disassembly of apoptotic Jurkat and PANX1 DN mutant expressing Jurkat cells. (b) Comparison of a selected panel of compounds from the LOPAC1280 library in modulating the formation of apoptotic bodies from apoptotic Jurkat and PANX1 DN mutant expressing Jurkat cells.(c) Sertraline reduces apoptotic body formation by apoptotic Jurkat cells under conditions when PANX1 channels are blocked. (d) Dose-dependent inhibition of apoptotic body formation by sertraline (n=3). Sertraline does not interfere with TO-PRO-3 uptake by apoptotic cells or ATP release into the supernatant under conditions when PANX1 channels are blocked (n=3).(e) Left, time-lapse images monitoring membrane blebbing of apoptotic Jurkat cells treated with or without sertraline or cytochalasin D (Cyto-D). Right, percentage of apoptotic Jurkat cells (based on cell rounding morphology) that has undergone membrane blebbing during 4-h time-lapse imaging (n=3).(f) Left, representative 4-h time-lapse images monitoring beaded apoptopodia formation by Jurkat cells treated with or without sertraline. Right, percentage of apoptotic Jurkat cells forming beaded apoptopodia (n=3). Sertraline inhibits the formation of apoptotic bodies(g) and beaded apoptopodia(h) by apoptotic THP-1 cells (n=3).(i) Vesicle transport inhibitor monensin reduces apoptotic body formation but not TO-PRO-3 uptake by apoptotic Jurkat cells under conditions when PANX1 channels are blocked (n=3). (j) Monensin does not interfere with membrane blebbing during apoptosis progression (n=3). (k) Monensin inhibits beaded-apoptopodia formation by Jurkat and THP-1 cells (n=3). (f,k) Actomyosin contraction and PANX1 channels are inhibited to promote the formation of beaded-apoptopodia in Jurkat cells. Error bars represent s.e.m. (c–k) Data are representative of at least two independent experiments. *P<0.05, **P<0.01, ***P<0.001, NS=P>0.5, unpaired Student's two-tailedt-test.
References
- Poon I. K., Hulett M. D. & Parish C. R. Molecular mechanisms of late apoptotic/necrotic cell clearance. Cell Death Differ. 17, 381–397 (2010). - PubMed
- Witasp E. et al. Bridge over troubled water: milk fat globule epidermal growth factor 8 promotes human monocyte-derived macrophage clearance of non-blebbing phosphatidylserine-positive target cells. Cell Death Differ. 14, 1063–1065 (2007). - PubMed
- Rubartelli A., Poggi A. & Zocchi M. R. The selective engulfment of apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and requires intracellular and extracellular calcium. Eur. J. Immunol. 27, 1893–1900 (1997). - PubMed
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources