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).