3.7. Deposition of aluminium on the surface of unicellular and multicellular organisms.
The results reported in the previous sections suggest that, in our experimental context, aluminium and silicon affected the preservation of SBO, probably by rapid binding with certain biomolecules. In order to successfully resist decay, an organic tissue presumably should adsorb aluminium or silicon ions before it becomes considerably degraded. To do so, it must contain molecules that effectively bind aluminium or silicon. The best candidates for such biomolecules are cell adhesion molecules and related extracellular molecules (CAMs and ECMs), because they evolved to provide effective adhesion to different charged substrates. Such molecules are typically absent in most unicellular organisms. Thus, we hypothesized that dead multicellular organisms should deposit aluminium and silicon more efficiently than unicellular organisms. To test this hypothesis, we investigated the deposition of aluminium on the carcasses of some unicellular and multicellular organisms.
In this experiment, we used three model organisms: freshwater unicellular flagellate Euglena gracilis, freshwater spongeSpongilla lacustris (a primitive multicellular organism), and soil social amoeba Dictyostelium discoideum . While the two former organisms are different in all biochemical aspects, D. discoideumhas both unicellular and multicellular stages. Under certain environmental conditions unicellular amoebae gather to form a loose multicellular aggregate. This then transforms into a tight aggregate (mound), then into a motile “slug”, a culminant, and finally a well-differentiated spore-producing fruiting body. Thus, in D. discoideum , we have a convenient example of unicellular and multicellular organisms with essentially the same biochemistry except for a set of CAMs and ECMs which is expressed predominantly at multicellular stages (these molecules are essential for cell-to-cell adhesion).
After the incubation of the dead model organisms with the Al3+-containing solution and removal of unbound aluminium, cell-bound Al3+ was detected by fluorescent staining with lumogallion, a very sensitive dye for bound aluminium detection. Lumogallion-Al complex has strong fluorescence at 488 nm (green); thus, the intensity of green fluorescence allows for the comparison of the amount of bound aluminium between the samples.
The sponge showed a very clear response to incubation with Al3+ and subsequent lumogallion staining. Autofluorescence in the non-stained control was negligible, as well as the fluorescence in the lumogallion-stained Al-negative control. However, Al-incubated lumogallion-stained specimens emitted bright green fluorescence (Figure 9 A-C).
The cells of E. gracilis had moderate autofluorescense and relatively intense fluorescence in the Al-negative control. The incubation with aluminium did not add any intensiveness to the emission as observed under the confocal microscope (Figure 9 D-F). The confocal microscopy also allowed us to recognize that autofluorescence and emission in the Al-negative control was mostly localized in some intracellular structures (probably chloroplasts and vacuoles), but not on or near the cell surface (Figure 9 G-I). Importantly, in the cells incubated with Al3+, fluorescence of the outer pellicle was not more intense than in the Al-negative cells (in fact, both fluorescent signals were equally low). The results imply that aluminium did not accumulate on the dead cells of E. gracilis in the same fast and efficient way as on the cells of the sponge.
The cells of D. discoideum at multicellular stages (culminants and fruiting bodies) emitted brightly at 488 nm (Figure 9 O, P). The strong Al-lumogallion signal from the multicellular structures was clearly different from the weak autofluorescence signal of the fruiting body (Figure 9 K) and the weak emission of the Al-negative control (Figure 9 M). Single amoebae showed very low fluorescence in all cases (Figure 9 J, L, N).
The results imply that the multicellular stages of D. discoideumexpress some aluminium-binding molecules which are present on the cell surface and retain their affinity to aluminium after cell death; these molecules are absent at the unicellular stage. In the case of social amoebae, the most plausible candidates are indeed cell adhesion molecules and related extracellular molecules (CAMs and ECMs). Unicellular stages (single amoebae) do not express CAMs, while the progression of D. discoideum through its multicellular stages is accompanied by the expression of several cell surface CAMs (Coates, Harwood, 2001; Siu et al., 2011). Sponges are multicellular animals that possess a large set of CAMs and ECMs, while Euglenozoa lack them (Seymour et al., 2004; Fahey, Degnan, 2010).