Figure 1.
Different microscopic techniques reveal the morphology of S. domuncula spicules; (A) light microscopy, (B–G) SEM.
(A) A tylostyle, a monaxonal rod with a terminal knob (k) on one side and a pointed tip at the other end. B and C show the blunt end with the knob (k) and some broken spicules exhibiting the axial canal (ac). (D) A broken spicule displaying the bulgy material constituting the axial filament (af). (E) A developing spicule with a progressively growing tip; the central core shell (c) around the axial canal (ac), and the final silica shell (s). The surface (su) of the spicule is marked. (F) Mineral deposits on a growing spicule causing the granular surface of its core (c) and shell (s) regions. (G) A broken spicule, displaying the internal mineral core (c) surrounding the axial canal (ac), and the outer shell (s).
Figure 2.
TEM images of the axial canal of spicules in primmorphs at different developmental stages.
(A) The three major developmental phases during spicule formation: phase I: primordial spicule comprising a large axial canal (ac) which is surrounded by an organic cylinder enclosing vesicles (v). phase II: the spicule shows the siliceous mantel (si) surrounding the small axial canal (ac) devoid of a pronounced axial filament. phase III: such spicules have a small sized axial canal and a distinct axial filament (af); scl, sclerocyte. (B) Spicule in phase II. Membraneous structures can be resolved in the axial canal (ac) which is surrounded by the silica mantel (si). (C) Spicule between phases II and III showing in the axial canal a well developed axial filament (af) embedded in membranous structures, which is surrounded by the silica mantel (si) (D) Mature spicule with an axial filament (af) without any cellular structures. One surrounding sclerocyte is marked (scl). (E) Axial canal (ac), close to the apex of the spicule, comprising a homogenous granular material. (F) Spicule with an axial canal at phase II/III. The axial canal (ac) comprises a growing axial filament (af). (G) Intermediate spicule phase between II and III comprising an axial canal (ac) showing cellular structures with vesicles (v) and one axial filament (af).
Figure 3.
Longitudinal sections through spicules, showing the process of evagination of cells into the axial canals (ac) of the spicules, as elucidated by TEM.
(A and B) Intracellular onset of spicule (sp) formation in sclerocytes (scl). The growing spicules are surrounded by silicasomes (sis). (C) A developing axial filament (af), that possesses at its blunt end several filaments (fi). (D) A longitudinal section through a spicule with its axial canal (ac). The axial canal is surrounded by a silica mantel (si). The silica fragments, that occurred during cutting of the primmorphs, were partially removed. The axial canal is closed at the apex (ap) of the spicule, while it is open at the end that is associated with the sclerocyte (scl). There the growth zone of the spicule (gr) exists. (E and F) The middle part of that spicule shows in the axial canal (ac) many silicasomes (sis). Those vesicles are surrounded by a membrane, which we perceive as cell membrane (><). Furthermore, the intracellular space (ics) and the developing axial filament (af) are marked. In the extracellular space (ecs) within the axial canal (ac) a silicasome (sis) can be identified. These images were taken close to the apex of the axial canal (ac). (G) Axial canal at the apex (ac), comprising no membranous structures and no axial filament. (H and I) An axial filament (af) in the extracellular space within the axial canal (ac).
Figure 4.
EDX analysess were performed of cross sections through primmorphs displaying growing spicules (sp).
The sections were made in transversal (A and B) and in longitudinal orientation (C and D); SEM analyses. (E) EDX spectra from areas in the extra-spicular space (ex-s) [sp1], within the siliceous mantel of the spicule [sp2], and the region across the axial canal (ac) of one spicule [sp3]. The areas where the spectroscopic analysis were performed are marked in (B).
Figure 5.
The ultrastructure of the vesicles/silicasomes was analyzed by TEM.
Intra-spicular (in-s) and extra-spicular (ex-s) regions, comprising silicasomes (sis), were studied. (A to D) The intra-spicular silicasomes were found (A to C) densely packed within the cell extensions protruding into the axial canal (ac) and also (D) outside of the cell extensions. (D) One silicasome (sis), identified in the extracellular space within the axial canal (ac), was surrounded by the silica mantel (si). (E and F) Silicasomes found on the surface of the spicules, in the extra-spicular space (ex-s). (E) Some of the silicasomes had an electron-dense content; scl, sclerocyte. (F) In all silicasomes (sis) the membrane was perforated; some of the pores are marked (><).
Figure 6.
Immunostaining of cryosections through primmorphs of S. domuncula showed growing spicules (sp).
The 8-µm thick frozen sections were reacted with one of the following polyclonal antibodies; with anti-silicatein PAb-aSILIC_SUBDO (A and B), with anti-aquaporin (PoAb-aAQP_SUBDO) (C and D), or with anti-arginine kinase (PoAb-aAK_SUBDO). (E and F) In one control series, the slices were reacted with preimmune serum from an animal used for immunization with aquaporin (G and H). The immunocomplexes were visualized with red florescent light, while the corresponding DAPI patterns were recorded with blue fluorescence light. All size bars represent 100 µm.
Figure 7.
Immunogold labeling electron microscopy [TEM] of the axial canal of spicules.
Antibodies against silicatein (A to C), against aquaporin (D to F) and against arginine kinase (G to I) were used. (A to C) The silicatein antibodies reacted with the axial filament (af), which was surrounded by the silica mantel (si), while (D to F) the aquaporin antibodies recognized their antigens primarily at the rim of the axial canal (ac) towards the silica mantel (si). (G to I) The anti-arginine kinase antibodies reacted in a more scattered pattern with the antigen in the axial canal, primarily recognizing membranous structures. The size of all bars represents 1 µm.
Figure 8.
Ultrastructure and immunoelectron microscopy prove the specificity of the antibodies.
Sections through spicules were prepared and inspected by TEM analysis. Parallel specimens were reacted either with antibodies or with the preimmune serum kept from this immunization. (A and B) Silicatein: (A) reaction with PAb-aSILIC_SUBDO; (B) incubation with the corresponding pre-immune serum. (C and D) Aquaporin: (C) reaction with PoAb-aAQP_SUBDO; (D) corresponding pre-immune serum. (E and F) Arginine kinase: (E) reaction with PoAb-aAK_SUBDO; (F) corresponding pre-immune serum. The axial canal (ac), the axial filament (af) and the silica shell (si) as well as the intra-cellular space (ics) and the extra-cellular space (ecs) are marked.
Figure 9.
The scheme depicts spicule formation via bio-inorganic self-organization.
(A) The spicule (sp) synthesis starts intracellularly in sclerocytes (scl). The primordial spicules are associated with filaments (fi) which are assumed to participate in the extrusion of the growing spicule. This phase is dominated by the expression of silicatein that – at the later stage – is required for the formation of both the core and the shell cylinder of the siliceous mantel of the spicule. The newly formed silicatein molecules undergo fractal organization. (B) The primordial spicule is extruded and becomes associated in the extracellular space with sclerocytes (scl) which intracellularly form the silicasomes (sis). These organelles contain silicatein and silicate that are released into the extra-spicular space and cause bio-silica formation. (C) The growth of the spicule (sp) continues in two directions; axial elongation and appositional growth/thickening. The bio-silica formation is mediated by silicatein (sil) under the consumption of the substrate silicate (si). Growth of spicule is driven both longitudinally and (subsequently) radially along the cell protrusion. During this phase the cell extensions elongate by evagination. The core of the spicule mantel is formed by silicatein, existing in the axial canal, and the shell by silicatein layered onto the outer surface of the growing spicule. (D) Final completion of the size and form of the spicule. After termination the spicule disconnects from the sclerocyte (not shown in the scheme) and the hole is closed by bio-silica formation. The direction of cell movement is indicated with an arrow.