Fig 1.
Crystal cells typically contain a single crystal.
(a) Section of crystal cell by TEM shows void previously occupied by crystal (asterisk). Crystal void is surrounded by mitochondria. Flattened nucleus of crystal cell (n) is applied to the cell membrane. Sparse additional inclusions include light and dark vesicles and a Golgi apparatus (g). (b) Reconstruction of interior of crystal cell from 123 serial sections. Mitochondria (red) surround crystal (light blue). The crystal complex sits in the cup formed by the nucleus (blue), and a Golgi apparatus (green) flanks the nucleus. Light and dark vesicles in light and dark pink. Crystal cell silhouette is shown in gray. Abbreviations: g–Golgi apparatus; er–endoplasmic reticulum; n–nucleus. Scale bar 500 nm.
Fig 2.
Orientation of crystal cells and position of crystal.
(a) Cup shaped nuclei of two crystal cells are apparent in an en face view of the edge of an animal stained with Hoechst nuclear dye. Orientations of the cups are indicated by red arrows. Oval-shaped nuclei of epithelial cells are tightly packed near the edge of the animal (below). Volume rendering of a series of confocal optical sections was created with Leica 3D imaging software. (b) Map of the positions and orientations of the cup-shaped nuclei of crystal cells. Outline traces the edge of the animal. Orientations of the openings of the cups are indicated by red arrows in mouths of the cups. Most cup openings are oriented towards the perimeter. (c) Effect of gravity on the positions of crystals inside crystal cells. Insets show the positions of crystals (c) inside their crystal cell in an animal on a horizontal surface (left), or tilted vertically for 2 min (center) or 30 min (right). Graphs compares the proportion of crystal cells with crystal shifted up, down or in the center under the three conditions. Error bars are SEM. *—p<0.05. **—p<0.005. Abbreviations: c–crystal; n–crystal cell nucleus. Scale bars: a– 5 μm; b– 100 μm; c– 2 μm (insets).
Fig 3.
Crystal cells are contacted by fiber and epithelial cells.
(a) Crystal cell (c) is contacted by fiber cell process (fp, above) and the cell body of a fiber cell (f, below). The identities and relationships of the cells were established by reconstructing 123 serial sections acquired with backscatter SEM. Asterisk marks the tiny void at the tip of the missing crystal. Crystal cell gives rise to thin processes (white arrows) and as well as larger processes (black arrows). (b) Surface rendering derived from the reconstruction shows that the crystal cell (magenta) is contacted by two fiber cells and their processes (blue). Cyan areas outline areas of contact of the fiber cell with the crystal cell. Crystal cells have numerous thick and thin side branches (black and white arrows, respectively). Inset enlarges a fiber cell process contacting a side branch of the crystal cell. (c) Section from the same stack shows a fiber cell body lying under the crystal cell contains a cluster of dark mitochondria and paler organelles, forming the mitochondrial complex that is a hallmark of fiber cells. (d) Crystal cell (c) in thin section SEM (above) is in tight contact with fiber cell process (fp). Comparable fiber cell contact in TEM thin section (below), where no junctional structures are apparent along the contact zone. (e) Appositions between a crystal cells (c) and epithelial cells (e) in thin section SEM (top) and TEM (below). Arrow indicates filamentous material spanning the cleft between the cells. (f) Edge of animal stained with Hoechst (blue) and Phalloidin (green) viewed by confocal microscopy. Maximum intensity projection of three sequential 0.185 μm optical sections. Phalloidin staining shows abundant actin filaments lining the plasma membranes of two crystal cell (c) and in the epithelial cells (e) that contact each of them. Abbreviations: c–crystal cell; e–epithelial cell; f–fiber cell; fp–fiber cell process. Scale bars: a, c– 1 μm; d– 200 nm; e– 500 nm; f– 5 μm.
Fig 4.
Chemical composition and appearance of isolated crystals selected in TEM to be analyzed by X-ray (a) and by electron energy loss spectroscopy (EELS) (b). The x-ray spectrum (a) from a small crystal (c, by TEM, contrast reversed) shows a large calcium peak (red). Blue record corresponds to signal from the support film. EELS data were obtained from the area along the edge of the crystal (box in d, visualized by STEM (HAADF) image), shown enlarged in (e). Boxes in (e) represent two subzones, producing a spectrum from the crystal (Box 2) shown in red in (b) and another from Box 1, over the substrate, shown in blue in (b). Box 2 yields a spectrum characteristic of calcium carbonate (b, red record); the spectrum here shows C K, Ca L2,3, and O K edges, together with C K energy loss containing 1s → π* peak at 289.3 eV an (inset) 1s → σ* peak at 300.0 eV. Box 1, control, is located on support film giving spectrum characteristic of amorphous carbon (b, blue line). The whole EELS dataset from boxed region in (d) was used to generate calcium map from Ca L2,3 signal (f), carbonate (C = O) map from 1s → π* signal (g), and oxygen map from O K-edge signal (h). Scale bars: c– 600 nm; d– 1 μm; e– 100 nm.
Fig 5.
Abundance of crystal cells affects responses to changes in direction gravity.
Transmitted polarized light image reveals numerous crystals arrayed around the edge of a typical animal (a), and only two crystals in an atypical animal (b). Inset (b) shows enlarged view of a crystal. (c), (d) Comparison of tracks of individual animals on a vertical substrate over the course of two hours, beginning 5 min after they were tilted vertically. Green and red dots mark, respectively, the beginning and end of each track. Tracks of many animals with a typical complement of crystals (c) include frequent episodes of movement in the direction opposing gravity (10 of 17 animals), while animals with <8 crystals (d) move predominantly in the direction of gravity (17 of 20 animals). (e) Comparison of net displacement over time on a vertical surface of animals containing a typical complement of crystals (light gray line; N = 49) and animals containing <8 crystals (dark gray line, N = 33). Animals with a typical complement of crystals on average show only a small downward displacement over the course of two hours whereas animals containing <8 crystals descend at a rate of ~2 mm per hour. Error bars are SEM. Scale bars: a, b– 100 μm; c, d– 5 mm.
Fig 6.
How crystal cells may be activated when tilted.
Possible mechanism of selective crystal cell activation in Trichoplax upon being tilted vertically. Schematic cross-section of Trichoplax attached to horizontal (a) or vertical (b) substrates, and en face view of animal on a vertical substrate (c). Cup-shaped nuclei (blue) are directed to the edge of the animal, regardless of its orientation with respect to gravity, while crystals move under the influence of gravity. Crystals in animals on a horizontal surface (a) generally remain in the nuclear cup, but when the surface is tilted vertically (b), crystals on the down side of Trichoplax fall out of the cup (red outline). We postulate that pressure exerted by the crystal on the plasma membrane activates mechanosensitive receptors in the membrane, which in turn causes the crystal cell to transmit a signal (red brackets). En face view (c) illustrates the distribution of activated crystal cells (red outlines and brackets) in an animal on a vertical substrate.