Paramecium, a Model to Study Ciliary Beating and Ciliogenesis: Insights From Cutting-Edge Approaches

Abstract

Cilia are ubiquitous and highly conserved extensions that endow the cell with motility and sensory functions. They were present in the first eukaryotes and conserved throughout evolution (Carvalho-Santos et al., 2011). Paramecium has around 4,000 motile cilia on its surface arranged in longitudinal rows, beating in waves to ensure movement and feeding. As with cilia in other model organisms, direction and speed of Paramecium ciliary beating is under bioelectric control of ciliary ion channels. In multiciliated cells of metazoans as well as paramecia, the cilia become physically entrained to beat in metachronal waves. This ciliated organism, Paramecium, is an attractive model for multidisciplinary approaches to dissect the location, structure and function of ciliary ion channels and other proteins involved in ciliary beating. Swimming behavior also can be a read-out of the role of cilia in sensory signal transduction. A cilium emanates from a BB, structurally equivalent to the centriole anchored at the cell surface, and elongates an axoneme composed of microtubule doublets enclosed in a ciliary membrane contiguous with the plasma membrane. The connection between the BB and the axoneme constitutes the transition zone, which serves as a diffusion barrier between the intracellular space and the cilium, defining the ciliary compartment. Human pathologies affecting cilia structure or function, are called ciliopathies, which are caused by gene mutations. For that reason, the molecular mechanisms and structural aspects of cilia assembly and function are actively studied using a variety of model systems, ranging from unicellular organisms to metazoa. In this review, we will highlight the use of Paramecium as a model to decipher ciliary beating mechanisms as well as high resolution insights into BB structure and anchoring. We will show that study of cilia in Paramecium promotes our understanding of cilia formation and function. In addition, we demonstrate that Paramecium could be a useful tool to validate candidate genes for ciliopathies.

Keywords: basal body; cilia; ciliary beating; cryo-tomography; ion channel; paramecium; transition zone.

FIGURE 1

(A) An image based on the sketch of the stages of the avoiding reaction drawn by Jennings (Jennings, 1906). Anterior mechanical stimulation by a cell swimming into an object leads to depolarization, opening of the Ca_V channels of the cilia, movement of the cell backward for a short time, twirling in place, and forward movement in a new direction. From Eckert (Eckert, 1972). (B) Scanning electron micrograph image of Paramecium showing metachronal waves of cilia (Courtesy of M. S. Valentine.).

FIGURE 2

(A) Basal body organization in Paramecium tetraurelia. Basal bodies (BB) are decorated by monoclonal anti-glutamylated tubulin ID5 antibodies (green). Note that in the anterior part of the cell (area 1 corresponding to the invariant field, traced in white), basal bodies are organized in doublets. In the posterior part of the cell, singlet basal bodies are observed (area 3). In the rest of the cell, doublets or singlets of basal bodies are observed (mixed field, area 2). Scale bar: 10 µm. (B,C) Immunofluorescence showing basal bodies decorated by ID5 antibodies (green) and cilia stained by anti-polyglutamylated tubulin (red). Transverse section showing that in the invariant field (area 1), each BB, which are organized in doublets, grow a cilium. Scale bar: 1 µm. (D) Schematic representation of the different steps (A–E) of BB duplication. (E) Longitudinal section of a ciliated basal body observed by transmission electron microscopy Scale bar: 200 nm. The arrows indicated the position of the three layers of the TZ. Terminal plate (T), intermediate plate (I) and axosomal plate (A). The dotted lines indicate the position of the transition fibers (1) and Y-links (2). (F) Schema of the BB appendages in a 2BB unit. The striated rootlet (SR) courses toward the anterior of the cell while the post-ciliary rootlet (Pc) is located near the 8th and 9th microtubule triplets. Also shown are the transverse rootlets on the anterior BB (Ta) and the posterior BB (Tp). (G) 3D representation showing the topographical relationships of BB and its anterior appendages: the striated ciliary rootlet (SR), the transverse microtubules (T) and the anterior left filament (ALF). The new BB (green disc). Reproduced/adapted with permission Aubusson et al, 2012. Journal of cell science, 125, 4395-4404 from Jerka-Diadosz et al., 2013.

FIGURE 3

These images illustrate (A) that the resting membrane potential of Paramecium is negative; the ciliary beat is toward the posterior and cell swims forward. (B) In depolarizing conditions, such as high K or Ba solutions, the cell’s membrane depolarizes and reaches threshold for the action potential, during which Ca²⁺ enters the cilium through Ca_V channels and the Ca²⁺ changes the power stroke toward the anterior, moving the cell backward. The action potential is quickly terminated, returned to resting V_m levels, and the extra Ca²⁺ removed (Kung et al., 1975).

FIGURE 4

Immunofluorescence and RNAi allow us to follow the location of channels and other proteins in the cell. (A) immunofluorescence allowed us to localize the Sk1a channel (FLAG-Sk1a, red) to mainly the cilia of Paramecium (green staining shows the GPI-anchored folate binding protein, FBP). Upon feeding BBS8 RNAi, the FLAG-SK1a disappears from the cilia, showing the dependence of this channel on the BBSome for trafficking. The GPI-anchored FBP remains undisturbed. Similarly (B) cells expressing PKD2-FLAG show the Pkd2 channel at the cell surface and in the cilia (red; basal bodies are green, stained with anti-Tetrahymena centrin) and after feeding the cells RNAi bacteria to deplete BBS8, the PKD2-FLAG channel is absent from the cilia. We have also used expressing cells and RNAi to examine the dependence of interacting proteins on one another for trafficking (C) Cells expressing PKD2-FLAG depleted in XNTA show no change in the localization of the Pkd2 channel and visa versa; cells expressing XNTA-FLAG depleted in PKD2 show no change in the localization of the XntA protein. Scale bars represent 15 µm (A) and (B) from Valentine et al., 2012 reproduced with changes with permission using Creative Commons CC BY license (C) reproduced with permissions from Valentine et al., 2019 with permission using Creative Commons CC BY license.

FIGURE 5

Depletion of SR gene products can lead to basal body and row misalignment. (A) Control cell (left) and RNAi treated cell with reduced SR proteins. (B) with basal bodies identified in green with ID5 antibodies, and the SRs identified in red with anti-SR antibody. Note misalignment of basal body rows and mis-orientation of basal bodies. (C) Control cells and (D) RNAi treated cells with reduced SR proteins as they swim in a pool of resting buffer for the same period of time. Note that the mis-orientation of basal bodies and rows leads to abnormal swimming (Nabi et al., 2019).

FIGURE 6

Basal body organization in Paramecium tetraurelia. (A) left: Paramecium stained Basal bodies (BB) are decorated by polyclonal anti-glutamylated tubulin (green) and anti-epiplasmin antibodies (red). Right: purified Paramecium cortex stained with anti-glutamylated tubulin (green) and anti-epiplasmin antibodies (red). Scale bar: 10 µm. (B) Purified basal body units obtained by 5 s of sonication. Scale bar: 2 µm. (C) Cryo-electron tomography of two Paramecium basal bodies. Z slice from a tomographic three-dimensional reconstruction showing a longitudinal section of a two basal body-cortical unit with the transition zone (green square), the central region (red square) and the proximal region (blue square). 1, 2, 3: Transverse projection of the cryo-tomogram at the respective three levels; 1′, 2′, 3’: 9-fold circularizations have been applied to the transverse projection. 1-1′, show the transition zone, 2-2′ show the central region of the basal body and 3-3′ show the basal body proximal region with the cartwheel structure inside. (D) The inner scaffold forms a dense helical lattice. Three-dimensional (3D) view of the ninefold symmetric central regions from Paramecium BB. Unrolled inner scaffold structures. In Paramecium a 2-start helix is observed (the pink color indicates one helix while the orange one indicated the second helix). Adapted from Le Guennec et al., 2020. (E) Cartoon of a longitudinal section of a Basal body. The microtubules are in grey, the cartwheel is in blue, CID in red, pinheads in violet, (A–C) linker in turquoise, triplet base in green and the inner scaffold in orange [Reprinted from (Klena et al., 2020; Le Guennec et al., 2020)]. The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 License (http://creativecommons.org/licenses/by-nc/4.0/).

FIGURE 7

Paramecium, a model to study basal body anchoring defects. (A) A1-A4: Control Paramecium (A1, A3) and FOPNL-depleted Paramecium (A2, A4) decorated for basal bodies (ID5 antibodies) observed by confocal at the cell cortex (A1, A3) or at intracytoplasmic level (A2, A4). Note that FOPNL depletion leads to a disorganized pattern of basal bodies at the cell cortex. Unanchored basal bodies are found in the cytoplasm (A4) (reproduced/adapted with permission "Aubusson et al, 2012. Journal of cell science, 125, 4395-440"). (B) Transmission electron microscopic images of a basal body doublet anchored at the cell surface. Note that the TZ vary in height between the ciliated basal body and the unciliated one (B1). B2-B4: Depletion of Centrin2 (B2), OFD1 (B3) (reprint from Bengueddach et al, 2017, Cilia 6, 6. doi:10.1186/s13630-017-0050-z), and FOPNL (B4) lead to unanchored basal bodies with defective organization of its distal end (reproduced/adapted with permission Aubusson et al, 2012. Journal of cell science, 125, 4395-4404). B5-B6: immunofluorescence of a control Paramecium (B5), or cell depleted for SF-assemblin depleted group-I proteins (B6) from Nabi et al., 2019 with permission, stained for the kinetodesmal fiber (red) and microtubule rootlets (green). Note the disorganization of the basal bodies and mis-orientation of their rootlets at the cell surface (Nabi et al., 2019). Depletion of Vfl3 protein (B7) induces the formation of more than one kinetodesmal fiber indicating rotational polarity defect (reprint from Bengueddach et al, 2017, Cilia 6, 6. doi:10.1186/s13630-017-0050-z). By contrast, the distal end of the basal body is well organized as after depletion of Centrin3 (B9) (reproduced/adapted with permission Aubusson et al, 2012. Journal of cell science, 125, 4395-4404). Scale bars: 250 nm.

FIGURE 8

(A) Paramecia expressing different TZ proteins fused with GFP. Cells were permeabilized before proceeding to immunostaining by ID5 (decorating BB and cilia in magenta) and a polyclonal anti-GFP (in green). Left panel: surface view of a Paramecium expressing TMEM216-GFP. Ciliated basal bodies of the invariant field (encircled in white) are stained by both GFP antibodies and ID5. In the other part of the cells, some basal bodies are labelled only by ID5. Right panels: confocal Z projections of ciliary rows, at the cell margin from TZ protein transformants. TMEM107-GFP, TMEM216-GFP, CEP290-GFP, and RPGRIP1L-GFP are localized only on the distal part of ciliated BBs. In addition, NPHP4-GFP can be observed at the BB proximal part. Note that ID5 antibodies better recognize short cilia. Bars = 10 and 1 μm. (B) Representative STED images revealing distinct localization patterns of several GFP-tagged TZ proteins. Cells were labelled with anti-GFP or ID5 (tubulin). A single ring differing in diameter is observed according to the observed protein. The mean diameters (distance between intensity maxima) and the number of BBs analyzed are given beneath each image, 2 replicates. (C) Graph (left) showing the mean diameter ± Standard Deviation of each toroid labeled by each GFP tagged TZ protein (see (B). Top right, schema representing the relative position of all toroids with respect to the position of tubulin and the ciliary membrane. Each TZ protein is shown in a different color reproduced from Gogendeau et al, 2020, PLoS Biol 18(3): e3000640. 10.1371/journal.pbio.3000640.