Fig 1.
3D cellular architecture of choanoflagellates and collar cells across the Choanozoa.
(A) Phylogenetic distribution of collar cells across the Choanozoa (Choanoflagellata + Metazoa [1,5]) showing the presence (black circle), absence (white circle), and putative losses (brown cross) of collar cells across lineages. The origin of collar cells is marked by the orange circle. Adapted from [1]. *Some lineages within the Bilateria have secondarily lost collar cells. (B) The choanoflagellate S. rosetta exhibits a complex life cycle, transitioning through both single and colonial collar cell types. The development of rosette colonies can be induced by RIF. Choanoflagellate colonies form through cytokinesis. (C–D) Characterisation of major organelles in S. rosetta labelled with fluorescent vital dyes (C) and by immunofluorescence (D). Arrowhead indicates nucleus of choanoflagellates cell; asterisks indicate the stained nucleoids of engulfed prey bacteria. Scale bar = 1 μm. (E–L) 3D ssTEM reconstruction of three single (S1–3) and three colonial (C1–3) S. rosetta cells (E, F). The association of the three colonial cells in context with each other are shown in the white box. The plasma membrane was made transparent (G, J), and glycogen and ER were removed to allow better visualisation of subcellular structures (H, K) and vesicle populations (I, L). Shown are apical vesicles (pink), food vacuoles (green), endocytotic vacuoles (fuschia), ER (yellow), extracellular vesicles (grey), filopodia (external, purple), flagellar basal body (light blue), flagellum (dark green), glycogen storage (white), Golgi apparatus and vesicles (purple), intercellular bridges (external, yellow; septa, red), large vesicles (brown), microvillar collar (light orange), mitochondria (red), nonflagellar basal body (dark orange), and nuclei (dark blue). Scale bar = approximately 1 μm (depending on position of structure along the z-axis). ER, endoplasmic reticulum; RIF, rosette-inducing factor; ssTEM, serial ultrathin transmission electron microscopy.
Fig 2.
3D ssTEM reconstructions allow for volumetric and numerical comparison of high-resolution single and colonial S. rosetta cells.
Shown are the mean volumetric breakdowns of three single (A) and three colonial (B) S. rosetta cells (left) and a generalised diagram of cell type ultrastructure (right). Colours are as in Fig 1. (C) Volumetric (%) (±SEM) (ER and endocytotic vacuoles) and numerical (μm−3) (±SEM) (endocytotic vacuoles, pseudopodia, Golgi-associated vesicles, and mitochondria) differences were found between single and colonial (n = 3) S. rosetta cells. *P < 0.05, **P < 0.01, ***P < 0.001. ER, endoplasmic reticulum; ssTEM, serial ultrathin transmission electron microscopy.
Fig 3.
Reconstructions of complete choanoflagellate RCs places colonial cells into context and unveils ultrastructural features involved in rosette formation and a novel cell type.
(A–D) 3D ssTEM reconstruction of a complete RC1. The plasma membrane was made transparent (B) to allow better visualisation of subcellular structures. Highlighted are contacting FP (C) and IBs (D). Cellular structures coloured as in Fig 1. Scale bar = approximately 1 μm. (E–L) 2D TEM and 3D ssTEM reconstructions of structures (*) differentially exhibited by colonial cells or involved in colony formation. Shown are the ER (E, F), IBs (G, H), EV (I, J), and FP (K, L). Scale bars = 200 nm. (M–P) Reconstruction of multiple S. rosetta colonies shows no strong pattern of volumetric distribution and bridge networks but reveals the presence of highly derived cell morphologies. (M) 3D ssTEM reconstructions of five complete rosettes (RC1–5) coloured by cell number (above), and 2D projections of bridge connections in 3D ssTEM reconstructions of RCs (below). Disconnected IBs marked by white arrowheads and lines. Asterisks mark the presence of highly derived cell morphologies in RC3 and RC4. Cells in RCs are numbered in order of their appearance along the z-axis. (N) Volumetric distribution of mean cell volumes (RC1–5) in RCs reveals no apparent pattern of cell distribution across the z-axis. (O, P). Two highly derived cell types, the ‘carrot cell’ (O) from RC3 and the ‘chili cell’ (P) from RC4, were identified in RCs. Colours as in Fig 1. Scalebar = approximately 1 μm. (Q–U) IBs in colonial S. rosetta exhibit a high diversity of morphologies, suggestive of disconnection. In addition to prior descriptions of IBs (arrowheads) and electron-dense septa (asterisks), bridges in colonial S. rosetta often display an asymmetrically distributed septum (Q), protracted and elongated morphology (R), disconnection from one of the contiguous cells (S), evidence of abscission (T), and putative inheritance of the septum (U). Scale bar = 200 nm. ER, endoplasmic reticulum; EV, endocytotic vacuoles; FP, filopodia; IB, intercellular bridge; RC, rosette colony; ssTEM, serial ultrathin transmission electron microscopy.
Fig 4.
3D cellular architecture of sponge choanocytes.
(A) Choanocytes line interconnected chambers in members of the Porifera and serve as feeding cells. (B) Mean volumetric breakdown of five sponge choanocytes. Colours are as in Fig 1. (C–E) 3D ssTEM of a section of choanocyte chamber containing five complete cells (B). The plasma membrane was rendered transparent (D), and food vacuoles and ER were removed to allow better visualisation of subcellular structures (E). Colours are as in Fig 1. Scale bar = approximately 1 μm. (F–G) Reconstruction and comparison of the sponge choanocyte (F) and choanoflagellate (G) apical poles shows distinct differences between the two cell types. Shown in the choanocyte reconstruction are the basal foot (red, associated with basal body), food vacuole (light green), ER (yellow), flagellar basal body (light blue), flagellum (dark green), Golgi apparatus and Golgi-associated vesicles (purple), microtubules (grey), mitochondria (red), nonflagellar basal body (dark orange), Type 1 vesicles (light orange), and Type 2 vesicles. Shown in the choanoflagellate reconstruction are the apical vesicles (pink), food vacuole (light green), ER (yellow), flagellar basal body (light blue), flagellum (dark green), Golgi apparatus and Golgi-associated vesicles (purple), glycogen (white), large vesicles (brown), microtubules (grey), microtubular ring (red), and nonflagellar basal body (dark orange). Scale bars = 200 nm. Diagrams of the choanocyte fine kinetid (F) and choanoflagellate fine kinetid (G) structure highlight the distinct differences. ER, endoplasmic reticulum; ssTEM, serial ultrathin transmission electron microscopy.