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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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Cellulose shell appears at the time of, but is not required for, cell rounding. (a) HMX44A cells 14 h after addition of DIF-1, labeled with Calcofluor. Top, phase contrast; bottom, fluorescence of the same microscopic field. Several round cells are Calcofluor-positive, several paddle cells are not. (b) An HMX44A cell 26 h after addition of DIF-1 showing an opening in its cellulose shell. Left, phase contrast; right, Calcofluor fluorescence. (c) HMX44A wild-type cells (left) and the corresponding cellulose-synthase–deficient (dcsA−) cells (right) were incubated in SB and DIF-1 for 47 h, then examined by phase-contrast microscopy (top) or after Calcofluor staining (middle). Also, they were compared as to their clonogenic ability (bottom). The percentage of cells able to regrow on addition of rich medium is indicated as a function of the duration of their initial incubation in SB alone (SB), or in SB plus DIF-1 (DIF). Altogether, the genetic removal of the cellulose shell from Dictyostelium cells alters neither other morphological signs of death nor the efficiency of clonogenic death.
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fig5: Cellulose shell appears at the time of, but is not required for, cell rounding. (a) HMX44A cells 14 h after addition of DIF-1, labeled with Calcofluor. Top, phase contrast; bottom, fluorescence of the same microscopic field. Several round cells are Calcofluor-positive, several paddle cells are not. (b) An HMX44A cell 26 h after addition of DIF-1 showing an opening in its cellulose shell. Left, phase contrast; right, Calcofluor fluorescence. (c) HMX44A wild-type cells (left) and the corresponding cellulose-synthase–deficient (dcsA−) cells (right) were incubated in SB and DIF-1 for 47 h, then examined by phase-contrast microscopy (top) or after Calcofluor staining (middle). Also, they were compared as to their clonogenic ability (bottom). The percentage of cells able to regrow on addition of rich medium is indicated as a function of the duration of their initial incubation in SB alone (SB), or in SB plus DIF-1 (DIF). Altogether, the genetic removal of the cellulose shell from Dictyostelium cells alters neither other morphological signs of death nor the efficiency of clonogenic death.

Mentions: Videos also show additional morphological features of this pathway. Thus, quite often, cells rounding up and vacuolizing could be seen to expel material (Videos 8–10; Arnoult et al., 2001), perhaps because of the increase in intracellular pressure due to the accumulation of fluid within the vacuoles. These extrusions could be interpreted as bursting out of cytoplasmic material through the outer cell layers. These include the peripheral actin rim, whose weakening might have consequences somewhat similar to blebbing in apoptotic animal cells, and the neo-synthesized cellulose shell, through which openings do occur (see Fig. 5 b). Also, a cell often initially presents several vacuoles, of which smaller ones empty into larger ones (Videos 9 and 11).


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)

Cellulose shell appears at the time of, but is not required for, cell rounding. (a) HMX44A cells 14 h after addition of DIF-1, labeled with Calcofluor. Top, phase contrast; bottom, fluorescence of the same microscopic field. Several round cells are Calcofluor-positive, several paddle cells are not. (b) An HMX44A cell 26 h after addition of DIF-1 showing an opening in its cellulose shell. Left, phase contrast; right, Calcofluor fluorescence. (c) HMX44A wild-type cells (left) and the corresponding cellulose-synthase–deficient (dcsA−) cells (right) were incubated in SB and DIF-1 for 47 h, then examined by phase-contrast microscopy (top) or after Calcofluor staining (middle). Also, they were compared as to their clonogenic ability (bottom). The percentage of cells able to regrow on addition of rich medium is indicated as a function of the duration of their initial incubation in SB alone (SB), or in SB plus DIF-1 (DIF). Altogether, the genetic removal of the cellulose shell from Dictyostelium cells alters neither other morphological signs of death nor the efficiency of clonogenic death.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Cellulose shell appears at the time of, but is not required for, cell rounding. (a) HMX44A cells 14 h after addition of DIF-1, labeled with Calcofluor. Top, phase contrast; bottom, fluorescence of the same microscopic field. Several round cells are Calcofluor-positive, several paddle cells are not. (b) An HMX44A cell 26 h after addition of DIF-1 showing an opening in its cellulose shell. Left, phase contrast; right, Calcofluor fluorescence. (c) HMX44A wild-type cells (left) and the corresponding cellulose-synthase–deficient (dcsA−) cells (right) were incubated in SB and DIF-1 for 47 h, then examined by phase-contrast microscopy (top) or after Calcofluor staining (middle). Also, they were compared as to their clonogenic ability (bottom). The percentage of cells able to regrow on addition of rich medium is indicated as a function of the duration of their initial incubation in SB alone (SB), or in SB plus DIF-1 (DIF). Altogether, the genetic removal of the cellulose shell from Dictyostelium cells alters neither other morphological signs of death nor the efficiency of clonogenic death.
Mentions: Videos also show additional morphological features of this pathway. Thus, quite often, cells rounding up and vacuolizing could be seen to expel material (Videos 8–10; Arnoult et al., 2001), perhaps because of the increase in intracellular pressure due to the accumulation of fluid within the vacuoles. These extrusions could be interpreted as bursting out of cytoplasmic material through the outer cell layers. These include the peripheral actin rim, whose weakening might have consequences somewhat similar to blebbing in apoptotic animal cells, and the neo-synthesized cellulose shell, through which openings do occur (see Fig. 5 b). Also, a cell often initially presents several vacuoles, of which smaller ones empty into larger ones (Videos 9 and 11).

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