Reorganization of complex ciliary flows around regenerating Stentor coeruleus

Abstract

The phenomenon of ciliary coordination has garnered increasing attention in recent decades and multiple theories have been proposed to explain its occurrence in different biological systems. While hydrodynamic interactions are thought to dictate the large-scale coordinated activity of epithelial cilia for fluid transport, it is rather basal coupling that accounts for synchronous swimming gaits in model microeukaryotes such as Chlamydomonas. Unicellular ciliates present a fascinating yet understudied context in which coordination is found to persist in ciliary arrays positioned across millimetre scales on the same cell. Here, we focus on the ciliate Stentor coeruleus, chosen for its large size, complex ciliary organization, and capacity for cellular regeneration. These large protists exhibit ciliary differentiation between cortical rows of short body cilia used for swimming, and an anterior ring of longer, fused cilia called the membranellar band (MB). The oral cilia in the MB beat metachronously to produce strong feeding currents. Remarkably, upon injury, the MB can be shed and regenerated de novo. Here, we follow and track this developmental sequence in its entirety to elucidate the emergence of coordinated ciliary beating: from band formation, elongation, curling and final migration towards the cell anterior. We reveal a complex interplay between hydrodynamics and ciliary restructuring in Stentor, and highlight for the first time the importance of a ring-like topology for achieving long-range metachronism in ciliated structures. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Keywords: Stentor; cilia coordination; ciliary flows; metachronal waves; morphogenesis; regeneration.

Figure 1.

(a) Schematic of a single Stentor with key morphological features highlighted. The membranellar band [8] comprises rows of oral cilia arranged in parallel stacks (each approx. 7.5 µm × 1.5 µm). (b) Confocal immunofluorescence images highlighting the structure and organization of the membranellar band (MB), cortical striation patterns and associated rows of short body cilia. Note the abrupt change in width at the locus of stripe contrast (LSC). (c) Top view of the frontal field (FF) and gullet region (G). Antibody used in (b,c) was anti-α-tubulin (scalebars = 10 µm).

Figure 2.

(a) Chronology of MB and flow restructuring during oral regeneration in Stentor coeruleus (see main text for details). At time 0, an existing MB was induced to shed by sucrose shock. After 1–2 h, a rift opens at the locus of stripe contrast. Between 2 and 5 h, oral cilia sprout, lengthen and eventually rearrange themselves into rows of stacked membranelles. After 5 h, the MB elongates and gradually migrates to assume a nearly circular structure at the anterior end. (b–g) particle image velocimetry (PIV) measurements of the extracellular flow fields associated with the regenerating MB for a control cell, and at the indicated times post sucrose shock for different regenerating individuals. (Colourmaps indicate flow speed, which changes significantly during regeneration. Brightfield images of the adhered organisms have been overlaid as masks on top of the flow maps. Black arrows label the MB location—wherever it is clearly identifiable.)

Figure 3.

Ciliary coordination over the course of oral regeneration. (a) A motion heatmap was used to localize the beating cilia, e.g. to a narrow band on the surface of the organism (arrow in the direction of increasing arclength from posterior to anterior). A region of interest parallel to the ciliary band (red), was used to generate intensity kymographs. Inset: kymograph reveals local image structure and coherence. (b) The 2D intensity autocorrelation shows increasing ciliary coordination over the course of regeneration. Sustained MCWs (parallel lines of high correlation in P–A direction) only emerge once MB regeneration has been almost completed. Here, the slope of the parallel lines equals MCW speed. Labels indicate time post sucrose shock.

Figure 4.

Correlating TEM sections with live-cell DIC microscopy at the same regeneration stage—at 6 h 45 min post sucrose-shock. (a) The ultrastructure is indistinguishable from control Stentor, fibrillar structures extend from the membranelles into the cytoplasm, in addition to transverse connections between neighbouring membranelles (arrows). (b) Cilia at the corresponding stage in live cells exhibit local but not global coordination—transient waves are propagated from the new oral primordium situated at the posterior (P), to the anterior end of the organism (A).