Resistance of Dictyostelium discoideum membranes to saponin permeabilization
© Mercanti et al; licensee BioMed Central Ltd. 2010
Received: 1 February 2010
Accepted: 28 April 2010
Published: 28 April 2010
Saponin is a mild detergent commonly used to permeabilize cells prior to immunofluorescence labeling of intracellular proteins. It has previously been used to that effect in Dictyostelium discoideum amoebae.
We show that saponin, contrary to Triton X-100 or alcohol, permeabilizes at best incompletely membranes of Dictyostelium. In cells exposed to osmotic stress, almost complete resistance to saponin permeabilization was observed.
Saponin should be used with special care to permeabilize Dictyostelium membranes. This unsusual property is presumably linked to the specific sterol composition of Dictyostelium membranes. It may also represent an adaptation of Dictyostelium to harsh conditions or to natural compounds encountered in its natural environment.
The localization of individual proteins in eukaryotic cells is often determined by immunofluorescence, a procedure requiring fixation and permeabilization of the cells before antibodies can be applied. Permeabilization of fixed cells is usually achieved by extracting membrane lipids with detergents like saponin or Triton X-100, or with alcohol. Mild permeabilization procedures are often preferred, in the hope of preserving better the cellular architecture. In particular, saponin (at concentrations of 0.1 to 0.5%) is a widely used detergent that extracts cholesterol from membranes. It is notably less prone than non-ionic detergents like Triton X-100 or NP-40 to extract membrane proteins from cellular membranes . Since different cholesterol concentrations are found in various cellular membranes, saponin can be used at very low concentrations (below 0.02%) to selectively permeabilize only a few membrane compartments of mammalian cells (e.g. the plasma membrane but not the endoplasmic reticulum) .
Dictyostelium amoebae are widely used to study many facets of cellular biology, such as the organization and function of the endocytic pathway. We  and others [4–6] have used saponin permeabilization to perform immnofluorescence on fixed cells. In the course of our studies, we were brought to suspect that saponin permeabilization of Dictyostelium cells may be incomplete, particularly in starved cells undergoing multicellular development. In this study, we show that Dictyostelium cells exposed to various conditions may exhibit highly variable degrees of resistance to saponin permeabilization.
Cells and reagents
Dictyostelium discoideum DH1-10 cells  were grown at 21°C in HL5 medium (14.3 g l-1 peptone (Oxoid LTD, Basingstoke, Hampshire, UK), 7.15 g l-1 yeast extract (Brunschwig BD Difco, Basel, Switzerland), 18 g l-1 maltose (Fluka, Buchs, Switzerland), 3.6 mM Na2HPO4, and 3.6 mM KH2PO4, pH 6.7).
Saponin prepared from Quillaja bark was from Sigma, paraformaldehyde was from Applichem and Triton X-100 from Fluka. Mouse monoclonal antibodies against the p80 endosomal marker (H161) were described previously .
To assess the efficacy of saponin permeabilization, Dictyostelium cells (5 × 105) were deposited on a glass coverslip (20 × 20 mm) in fresh HL5 medium (2 ml), and allowed to attach at 21°C for 3 h, then fixed for 30 minutes in HL5 medium containing 4% paraformaldehyde. Fixed cells were rinsed two times with PBS, and permeabilized with saponin (0.5% in PBS for 15 minutes) or with Triton X-100 (0.1% in PBS for 3 minutes). Note that these concentrations are well above the critical micellar concentrations of saponin (0.1%) and Triton X-100 (0.02%). Permeabilized cells were pre-incubated for 15 minutes in PBS containing 0.2% BSA (PBS-BSA), then incubated for 1 hour in PBS-BSA containing an Alexa-488-coupled H161. Cells were washed three times in PBS-BSA, one time in PBS, and cellular lipids were extracted in methanol at -20°C for 2 minutes. Cells were then rinsed in PBS, pre-incubated for 15 minutes in PBS-BSA, and incubated for 1 hour in PBS-BSA containing H161 antibody coupled to Alexa-647. Finally, cells were washed three times with PBS-BSA, one time with PBS, and mounted in Mowiol . Stained cells were observed in a LSM510 confocal microscope (Zeiss) and pictures were taken consecutively with identical settings to allow direct comparison between the signal intensities observed in various conditions within the same experiment.
To test permeabilization under starvation conditions, the same procedure was followed, except that cells were incubated for 30 minutes in starvation buffer (SB: 2 mM Na2HPO4, 14.7 mM KH2PO4, pH 6.0) just before fixation. Alternatively, an isotonic starvation buffer (SB+100 mM sorbitol) was used when indicated.
Results and discussion
In addition to inducing starvation, SB buffer is strongly hypotonic. In order to test if resistance to saponin permeabilization was induced by starvation or by hypoosmotic conditions, we performed the same experiment with cells exposed to an isotonic starvation buffer (SB+100 mM sorbitol). In this medium, Dictyostelium cells are effectively starved, since sorbitol is not metabolized . Efficient saponin permeabilization was observed in these cells (Figure 2), suggesting that osmotic conditions, rather than starvation, modulate resistance of Dictyostelium membranes to saponin permeabilization.
Together these observations indicate that Dictyostelium membranes are intrinsically resistant to saponin permeabilization and that this resistance can be further enhanced by an osmotic shock. It seems likely that the resistance of Dictyostelium membranes to saponin reflects at least in part its specific sterol composition, that has been likened to that of plant cells . It remains to be established by which mechanism osmotic conditions can modulate resistance to saponin.
From a technical point of view, partial or complete resistance of Dictyostelium membranes to saponin permeabilization may cause a number of artifacts in immunofluorescence experiments. It may for example lead to underestimate the amount of intracellular protein relative to surface protein (compare Figure 1A and 1B), or to believe that some proteins are relocated to the cell surface in starved Dictyostelium cells (compare Figure 1A and 1C). On the other hand, in combination with appropriate controls, saponin permeabilization may provide a tool to study the peculiar properties of Dictyostelium membranes. While this study was focused on a marker of endocytic compartments, a similar approach may establish to what extent other cellular membranes are resistant to saponin extraction.
Finally, from an ecological perspective, saponins are synthesized by a variety of plants, and have been proposed to function as antimicrobial phytoprotectants . It seems plausible that Dictyostelium, a soil-dwelling amoeba, may be naturally exposed to plant saponins, and might benefit from being resistant to its detergent activities. Resistance of endosomal membranes to permeabilization may also limit escape of phagocytosed microorganisms from phagosomes, thereby preventing their access to the cell's cytosol.
This research was supported by grants to P.C. from the Swiss National Science Foundation, the Doerenkamp-Zbinden Foundation and the Fondation E. Naef pour la Recherche in Vitro (FENRIV).
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