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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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Diverse cell aspects along Dictyostelium terminal differentiation to cell death. (a) HMX44A Dictyostelium cells vegetatively growing in rich medium, or (b) after 8 h of incubation in starvation saline SB in the presence of an excess of cAMP. Many cells have acquired a flat morphology with a number of filopodia, and can keep this morphology for days on further incubation in SB medium. (c) The differentiation factor DIF-1 added to SB induces a sequence of other morphological changes. After 8 to 16 h, many cells acquire a paddle shape (d) that transforms into a round shape. (e) Round cells vacuolize, (f) then progressively, many of the vacuolated cells undergo membrane permeabilization and thus fluoresce red when incubated with propidium iodide, whereas cells with still intact membrane fluoresce green on incubation in a fluo-rescein diacetate solution. At 48 h, about half of the cells fluoresce red. (g) At 48 h of development, some stalks of fully developed AX2 fruiting bodies also show cells fluorescing red or green, often with an alternate pattern. (h) Approximate time scale of terminal differentiation and death, as reflected by sequential morphological changes, of HMX44A Dictyostelium cells subjected to starvation medium and receiving DIF-1 at time zero. There is marked heterogeneity between cells in terms of time to access a given aspect and duration of time with a given aspect.
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fig1: Diverse cell aspects along Dictyostelium terminal differentiation to cell death. (a) HMX44A Dictyostelium cells vegetatively growing in rich medium, or (b) after 8 h of incubation in starvation saline SB in the presence of an excess of cAMP. Many cells have acquired a flat morphology with a number of filopodia, and can keep this morphology for days on further incubation in SB medium. (c) The differentiation factor DIF-1 added to SB induces a sequence of other morphological changes. After 8 to 16 h, many cells acquire a paddle shape (d) that transforms into a round shape. (e) Round cells vacuolize, (f) then progressively, many of the vacuolated cells undergo membrane permeabilization and thus fluoresce red when incubated with propidium iodide, whereas cells with still intact membrane fluoresce green on incubation in a fluo-rescein diacetate solution. At 48 h, about half of the cells fluoresce red. (g) At 48 h of development, some stalks of fully developed AX2 fruiting bodies also show cells fluorescing red or green, often with an alternate pattern. (h) Approximate time scale of terminal differentiation and death, as reflected by sequential morphological changes, of HMX44A Dictyostelium cells subjected to starvation medium and receiving DIF-1 at time zero. There is marked heterogeneity between cells in terms of time to access a given aspect and duration of time with a given aspect.

Mentions: Vegetative cells growing in rich medium (Fig. 1 a and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200212104) were transferred to starvation medium SB containing 3 mM cAMP, in which they were incubated for 8 h. Control groups were then further incubated in SB alone, leading to a majority of cells with flat body and filopodia (Fig. 1 b and Video 2) that can survive as such for several days. In contrast, incubation in SB in the presence of DIF-1 led to the emergence within 8–16 h of very motile and strikingly compartmentalized cells that we call paddle cells (Fig. 1 c and Videos 3–7; see below). These stop moving within 15–26 h and change morphology to round cells (Fig. 1 d and Video 8). Round cells then progress toward massive vacuolization (Fig. 1 e and Videos 9–11) and still later to rupture of the cytoplasmic membrane (Video 12), which allows propidium iodide staining of the cell remnant (Cornillon et al., 1994). Incubation with propidium iodide leads to <50% of the cells fluorescing red after 2 d of starvation and DIF-1, whereas most of the other cells still stain green due to live-cell labeling with fluorescein diacetate (Fig. 1 f). Thus, more than two days are required in vitro (i.e., in submerged monolayers) for half of the cells to die, using this “morphological” criterion for cell death. In vivo (i.e., during normal development), developing Dictyostelium AX2 cells in stalks would also stain early with fluorescein diacetate and later with propidium iodide, although we found considerable interstalk variation in the timing of this shift in staining. After 2 d of starvation, stalks often contained areas of cells labeled with fluorescein diacetate alternating with areas of cells labeled with propidium iodide (Fig. 1 g). Dictyostelium developmental cell death in vivo thus includes late permeabilization to propidium iodide. Altogether, both in vivo in stalks and in vitro in monolayers, in terms of permeabilization of the plasma membrane death of 50% of the cells requires at least two days. Microscopic examination and numerous time-lapse movies (unpublished data) led to the establishment of an approximate time scale for these various cell aspects (Fig. 1 h), which often overlap within a cell population due to marked heterogeneity between cells as to the starting time and duration of a given stage.


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)

Diverse cell aspects along Dictyostelium terminal differentiation to cell death. (a) HMX44A Dictyostelium cells vegetatively growing in rich medium, or (b) after 8 h of incubation in starvation saline SB in the presence of an excess of cAMP. Many cells have acquired a flat morphology with a number of filopodia, and can keep this morphology for days on further incubation in SB medium. (c) The differentiation factor DIF-1 added to SB induces a sequence of other morphological changes. After 8 to 16 h, many cells acquire a paddle shape (d) that transforms into a round shape. (e) Round cells vacuolize, (f) then progressively, many of the vacuolated cells undergo membrane permeabilization and thus fluoresce red when incubated with propidium iodide, whereas cells with still intact membrane fluoresce green on incubation in a fluo-rescein diacetate solution. At 48 h, about half of the cells fluoresce red. (g) At 48 h of development, some stalks of fully developed AX2 fruiting bodies also show cells fluorescing red or green, often with an alternate pattern. (h) Approximate time scale of terminal differentiation and death, as reflected by sequential morphological changes, of HMX44A Dictyostelium cells subjected to starvation medium and receiving DIF-1 at time zero. There is marked heterogeneity between cells in terms of time to access a given aspect and duration of time with a given aspect.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172757&req=5

fig1: Diverse cell aspects along Dictyostelium terminal differentiation to cell death. (a) HMX44A Dictyostelium cells vegetatively growing in rich medium, or (b) after 8 h of incubation in starvation saline SB in the presence of an excess of cAMP. Many cells have acquired a flat morphology with a number of filopodia, and can keep this morphology for days on further incubation in SB medium. (c) The differentiation factor DIF-1 added to SB induces a sequence of other morphological changes. After 8 to 16 h, many cells acquire a paddle shape (d) that transforms into a round shape. (e) Round cells vacuolize, (f) then progressively, many of the vacuolated cells undergo membrane permeabilization and thus fluoresce red when incubated with propidium iodide, whereas cells with still intact membrane fluoresce green on incubation in a fluo-rescein diacetate solution. At 48 h, about half of the cells fluoresce red. (g) At 48 h of development, some stalks of fully developed AX2 fruiting bodies also show cells fluorescing red or green, often with an alternate pattern. (h) Approximate time scale of terminal differentiation and death, as reflected by sequential morphological changes, of HMX44A Dictyostelium cells subjected to starvation medium and receiving DIF-1 at time zero. There is marked heterogeneity between cells in terms of time to access a given aspect and duration of time with a given aspect.
Mentions: Vegetative cells growing in rich medium (Fig. 1 a and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200212104) were transferred to starvation medium SB containing 3 mM cAMP, in which they were incubated for 8 h. Control groups were then further incubated in SB alone, leading to a majority of cells with flat body and filopodia (Fig. 1 b and Video 2) that can survive as such for several days. In contrast, incubation in SB in the presence of DIF-1 led to the emergence within 8–16 h of very motile and strikingly compartmentalized cells that we call paddle cells (Fig. 1 c and Videos 3–7; see below). These stop moving within 15–26 h and change morphology to round cells (Fig. 1 d and Video 8). Round cells then progress toward massive vacuolization (Fig. 1 e and Videos 9–11) and still later to rupture of the cytoplasmic membrane (Video 12), which allows propidium iodide staining of the cell remnant (Cornillon et al., 1994). Incubation with propidium iodide leads to <50% of the cells fluorescing red after 2 d of starvation and DIF-1, whereas most of the other cells still stain green due to live-cell labeling with fluorescein diacetate (Fig. 1 f). Thus, more than two days are required in vitro (i.e., in submerged monolayers) for half of the cells to die, using this “morphological” criterion for cell death. In vivo (i.e., during normal development), developing Dictyostelium AX2 cells in stalks would also stain early with fluorescein diacetate and later with propidium iodide, although we found considerable interstalk variation in the timing of this shift in staining. After 2 d of starvation, stalks often contained areas of cells labeled with fluorescein diacetate alternating with areas of cells labeled with propidium iodide (Fig. 1 g). Dictyostelium developmental cell death in vivo thus includes late permeabilization to propidium iodide. Altogether, both in vivo in stalks and in vitro in monolayers, in terms of permeabilization of the plasma membrane death of 50% of the cells requires at least two days. Microscopic examination and numerous time-lapse movies (unpublished data) led to the establishment of an approximate time scale for these various cell aspects (Fig. 1 h), which often overlap within a cell population due to marked heterogeneity between cells as to the starting time and duration of a given stage.

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.

Show MeSH
Related in: MedlinePlus