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Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

Levraud JP, Adam M, Luciani MF, de Chastellier C, Blanton RL, Golstein P - J. Cell Biol. (2003)

Bottom Line: Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells.A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin.The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, INSERM/CNRS, Case 906, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France.

ABSTRACT
Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

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Paddle cells, emergence, and preliminary characterization. (a–c) Emergence from a cell clump at 9.5, 10, and 10.5 h, respectively, after addition of DIF-1. (d) Three paddle cells 15 h after addition of DIF-1. Phase contrast (left) or fluorescence microscopy (right) after double staining, with CMXRos labeling (red) the mitochondria in the organelle-rich posterior region of the paddle cells, and with phalloidin labeling (green) the actin-rich propodia. (e) HMX44A cells transfected with ecmA::GFP 16 h after addition of DIF-1, examined by phase contrast (top) or fluorescence (bottom). The paddle cell almost in the middle of the field is among the most fluorescent cells. (f) Two-phase contrast microscopy fields of Neutral Red–stained cells 16 h after addition of DIF-1, exemplifying the fact that most paddle cells are stained and most other cells are not.
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fig2: Paddle cells, emergence, and preliminary characterization. (a–c) Emergence from a cell clump at 9.5, 10, and 10.5 h, respectively, after addition of DIF-1. (d) Three paddle cells 15 h after addition of DIF-1. Phase contrast (left) or fluorescence microscopy (right) after double staining, with CMXRos labeling (red) the mitochondria in the organelle-rich posterior region of the paddle cells, and with phalloidin labeling (green) the actin-rich propodia. (e) HMX44A cells transfected with ecmA::GFP 16 h after addition of DIF-1, examined by phase contrast (top) or fluorescence (bottom). The paddle cell almost in the middle of the field is among the most fluorescent cells. (f) Two-phase contrast microscopy fields of Neutral Red–stained cells 16 h after addition of DIF-1, exemplifying the fact that most paddle cells are stained and most other cells are not.

Mentions: After addition of DIF-1, starved cells tend to form clumps, out of which paddle cells emerge at 8–16 h (Fig. 2, a–c). A few paddle cells can occasionally be seen in SB only, with no added DIF-1, perhaps related to endogenous production of small amounts of DIF-1 even by these HMX44A cells. Paddle cells morphologically comprise an anterior half (Fig. 2 d and Video 3) that we call propodium. The propodium by phase microscopy looks amorphous and opaque gray, and by fluorescence microscopy is stained by the F-actin–specific stain phalloidin (Fig. 2 d). In marked contrast, the posterior half of paddle cells is heterogenous and contains many of the cell organelles. This posterior half is stained by CMXRos (Fig. 2 d) or by MitoTracker® Green (not depicted) and thus bears mitochondria, and by Neutral Red (Fig. 2 f), a classical lysosomal stain. It also contains the DAPI-stainable nucleus (unpublished data). When a paddle cell evolves into a vacuolized cell, the Neutral Red label is found within the vacuole and progressively decreases in intensity (unpublished data). Contrary to other cells in the same preparations, not only are paddle cells heavily labeled by Neutral Red (Fig. 2 f), but also, in HMX44A cells transfected with GFP under an ecmA promoter, they show induction of GFP (Fig. 2 e), two characteristics of prestalk cells. Most interestingly, morphologically similar (if not identical) cells were identified in vivo in dissociated slug cell populations by Inouye's group (see Fig. 12 in Yoshida and Inouye, 2001). There is consistency between ecmA and Neutral Red positivity of paddle cells classifying them as prestalk cells, their position on a pathway leading to cell death, and the presence of morphologically very similar cells in slugs.


Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

Levraud JP, Adam M, Luciani MF, de Chastellier C, Blanton RL, Golstein P - J. Cell Biol. (2003)

Paddle cells, emergence, and preliminary characterization. (a–c) Emergence from a cell clump at 9.5, 10, and 10.5 h, respectively, after addition of DIF-1. (d) Three paddle cells 15 h after addition of DIF-1. Phase contrast (left) or fluorescence microscopy (right) after double staining, with CMXRos labeling (red) the mitochondria in the organelle-rich posterior region of the paddle cells, and with phalloidin labeling (green) the actin-rich propodia. (e) HMX44A cells transfected with ecmA::GFP 16 h after addition of DIF-1, examined by phase contrast (top) or fluorescence (bottom). The paddle cell almost in the middle of the field is among the most fluorescent cells. (f) Two-phase contrast microscopy fields of Neutral Red–stained cells 16 h after addition of DIF-1, exemplifying the fact that most paddle cells are stained and most other cells are not.
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Related In: Results  -  Collection

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fig2: Paddle cells, emergence, and preliminary characterization. (a–c) Emergence from a cell clump at 9.5, 10, and 10.5 h, respectively, after addition of DIF-1. (d) Three paddle cells 15 h after addition of DIF-1. Phase contrast (left) or fluorescence microscopy (right) after double staining, with CMXRos labeling (red) the mitochondria in the organelle-rich posterior region of the paddle cells, and with phalloidin labeling (green) the actin-rich propodia. (e) HMX44A cells transfected with ecmA::GFP 16 h after addition of DIF-1, examined by phase contrast (top) or fluorescence (bottom). The paddle cell almost in the middle of the field is among the most fluorescent cells. (f) Two-phase contrast microscopy fields of Neutral Red–stained cells 16 h after addition of DIF-1, exemplifying the fact that most paddle cells are stained and most other cells are not.
Mentions: After addition of DIF-1, starved cells tend to form clumps, out of which paddle cells emerge at 8–16 h (Fig. 2, a–c). A few paddle cells can occasionally be seen in SB only, with no added DIF-1, perhaps related to endogenous production of small amounts of DIF-1 even by these HMX44A cells. Paddle cells morphologically comprise an anterior half (Fig. 2 d and Video 3) that we call propodium. The propodium by phase microscopy looks amorphous and opaque gray, and by fluorescence microscopy is stained by the F-actin–specific stain phalloidin (Fig. 2 d). In marked contrast, the posterior half of paddle cells is heterogenous and contains many of the cell organelles. This posterior half is stained by CMXRos (Fig. 2 d) or by MitoTracker® Green (not depicted) and thus bears mitochondria, and by Neutral Red (Fig. 2 f), a classical lysosomal stain. It also contains the DAPI-stainable nucleus (unpublished data). When a paddle cell evolves into a vacuolized cell, the Neutral Red label is found within the vacuole and progressively decreases in intensity (unpublished data). Contrary to other cells in the same preparations, not only are paddle cells heavily labeled by Neutral Red (Fig. 2 f), but also, in HMX44A cells transfected with GFP under an ecmA promoter, they show induction of GFP (Fig. 2 e), two characteristics of prestalk cells. Most interestingly, morphologically similar (if not identical) cells were identified in vivo in dissociated slug cell populations by Inouye's group (see Fig. 12 in Yoshida and Inouye, 2001). There is consistency between ecmA and Neutral Red positivity of paddle cells classifying them as prestalk cells, their position on a pathway leading to cell death, and the presence of morphologically very similar cells in slugs.

Bottom Line: Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells.A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin.The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, INSERM/CNRS, Case 906, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France.

ABSTRACT
Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

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