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Review
. 2021 Sep 24;12(10):1493.
doi: 10.3390/genes12101493.

Using Paramecium as a Model for Ciliopathies

Megan Valentine  1 , Judith Van Houten  2
Affiliations
Review

Using Paramecium as a Model for Ciliopathies

Megan Valentine et al. Genes (Basel). .

Abstract

Paramecium has served as a model organism for the studies of many aspects of genetics and cell biology: non-Mendelian inheritance, genome duplication, genome rearrangements, and exocytosis, to name a few. However, the large number and patterning of cilia that cover its surface have inspired extraordinary ultrastructural work. Its swimming patterns inspired exquisite electrophysiological studies that led to a description of the bioelectric control of ciliary motion. A genetic dissection of swimming behavior moved the field toward the genes and gene products underlying ciliary function. With the advent of molecular technologies, it became clear that there was not only great conservation of ciliary structure but also of the genes coding for ciliary structure and function. It is this conservation and the legacy of past research that allow us to use Paramecium as a model for cilia and ciliary diseases called ciliopathies. However, there would be no compelling reason to study Paramecium as this model if there were no new insights into cilia and ciliopathies to be gained. In this review, we present studies that we believe will do this. For example, while the literature continues to state that immotile cilia are sensory and motile cilia are not, we will provide evidence that Paramecium cilia are clearly sensory. Other examples show that while a Paramecium protein is highly conserved it takes a different interacting partner or conducts a different ion than expected. Perhaps these exceptions will provoke new ideas about mammalian systems.

Keywords: Paramecium; cilia; ciliate; ciliopathy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 2
Figure 2
Subcellular localization of protein encoded by monogenic genes of nephronophthisis-related cilioipathies (NPHP-RC). Subcellular localization of proteins encoded by monogenic genes of NPHP-RC is depicted. Proteins are color-coded based on their respective disease group: Nephronphthisis (NPHP), Senior–Loken syndrome (SLS), Joubert syndrome (JBTS), Meckel–Gruber syndrome (MKS), Bardet–Biedl syndrome (BBS), and skeletal ciliopathies. It becomes apparent that disease groups cluster to distinct subcellular localizations. IFT, intraflagellar transport. Reproduced from [5], with permission.
Figure 3
Figure 3
Overview of the anatomy of the centrosome/cilium complex and its sub-compartments. This complex is composed of three compartments, the centriole, the cilium, and the centriolar satellites. In primary cilia, the centrosome, consisting of two microtubule-organizing centers, recruits pericentriolar material (PCM). In Paramecium, these structures are not present, but instead each cilium is anchored by a basal body. In both, the centrioles (or basal bodies) differ in age where one is called the mother (the older) and the younger, called the daughter. The mother has distal appendages, and for primary cilia, the mother also acts as the basal body for cilium assembly. The primary cilium itself is compartmentalized into different regions, including the transition zone (TZ), the axoneme, and the ciliary tip. (A) A cross section of the proximal end of the centrioles containing nine triplets of microtubules symmetrically arranged in a ring connected by A–C linkers. These triplets are connected to the inner core of the centriole by radial spokes. The PCM material is organized into concentric layers and those layers are overlayed by a filamentous material, pericentrin. (B) The transition zone (TZ) has microtubule pairs, as the outmost microtubule does not extend the length of the cilium. This region acts as a barrier that regulates proteins into and out of the cilium through the NPHP-MKS-JBTS module. Transition fibers help to anchor the basal body to the ciliary membrane. (C) The ciliary axoneme is the core of the primary cilium and lacks a central pair of microtubules (that are present in motile cilia). These outer doublets of microtubules act as roadways for the IFT-A and IFT-B complexes, as well as the BBSome, to move cargo along the cilium. IFT-B and anterograde transport relies on kinesin-2 (blue) motors while retrograde transport and IFT-A rely on cytoplasmic dynein-2 (red) motors. The BBSome complex interacts with the IFT particles the move cargo into and out of the cilium. (D) The ciliary tip is a specialized region of primary cilia. Here, IFT particles, Hedgehog pathway components, and other microtubule-associated proteins work to regulate IFT remodeling, the length of the cilium, and Hedgehog signaling. Reproduced from [19], with permission through CC BY license.
Figure 4
Figure 4
Structural and functional features of motile and sensory cilia are associated with ciliopathies. (a) the major structures of motile and non-motile cilia. (b) Major sites of action for ciliopathy-associated proteins that are components of motile cilia (motility apparatus or transcription factors required for the generation of motile cilia) and sensory cilia (axonemal and signaling proteins, ciliary tip proteins or inversin (INV) compartment proteins). The asterisks indicate proteins that are also localized to other ciliary regions during ciliogenesis or ciliary trafficking. Circled numbers indicate one or more ciliopathies that result from defects in the different ciliary compartments and proteins. (c) Ciliopathies grouped into major categories that are associated with proteins and ciliary regions in part B. Reproduced from [7], with permission.
Figure 5
Figure 5
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 CaV channels and the Ca2+ changes the power stroke toward the anterior, moving the cell backward. The action potential is quickly terminated, returned to resting Vm levels, and the extra Ca2+ removed. Reproduced from [20] with permission.
Figure 1
Figure 1
Line drawing from a scanning electron microscope image of P. tetraurelia. Courtesy of J. Van Houten and 1988 Grass Calendar.
Figure 6
Figure 6
Series of steps in the swimming path of a Paramecium cell that bumps into a solid object (upper right), reverses, pivots in place, and finally swims off in a new direction [44].
Figure 7
Figure 7
Immunofluorescence of FLAG-Xnt, an interacting partner of PKD2. Only the merged images are shown here. FLAG control is a cell microinjected with FLAG-pPXV vector and fed RNAi empty vector bacteria (not shown). FLAG-Xnt controls are cells expressing FLAG-Xnt and fed bacteria with an RNAi empty vector. “BBS8”cells are expressing FLAG-Xnt and are also depleted of BBS8 mRNA by RNAi. Cells were immunostained with anti-FLAG (red) and basal body (green for Tetrahymena centrin) antibodies. (Valentine unpublished).
Figure 8
Figure 8
Summary cartoons of trafficking of membrane proteins to the cilia. (A) shows the folate receptor with its fatty acid tail moving from the ER directly to the plasma membrane before diffusing into the ciliary membrane. The XNT protein emerges from the Golgi on vesicles and moves to the plasma membrane and then to the contiguous ciliary membrane. The protein channels SK1a and PKD2 also emerge from the Golgi on vesicles but these vesicles are guided by the BBSome past the TZ and into the ciliary membrane. The motor proteins dynein and kinesin move the IFT complexes up and down the cilium and deposit their cargoes in the cilium. (B) shows that the calcium channel and its putative partner the plasma membrane calcium pump move on vesicles from the Golgi to the ciliary membrane without aid of the BBSome. When they reach the ciliary membrane, they are transported past the TZ and into the cilium.
Figure 9
Figure 9
Immunofluorescence of FLAG-SK1a channels and folate chemoreceptor (FBP) a GPI anchored protein with antibodies against the FBP. Note that both are in the cilia, while FBP is in both the cilia and cell membrane. Only the merged images are shown here, but all controls are available [10]. FLAG control is a cell microinjected with FLAG-pPXV vector and fed RNAi empty vector bacteria. FLAG-SK1a control is a cell expressing FLAG-SK1a and fed bacteria with an RNAi empty vector. “BBS8”cells are expressing FLAG-Sk1a channel and are also depleted of BBS8 RNA by RNAi. Cells were immune stained with anti-FLAG (red) and anti-FBP (green) antibodies. These images are from a larger published study with more BBS genes silenced, and with similar results. Note that Sk1a is missing from cilia after silencing BBS8, but the FBP remains in the cilia. Reproduced from [8], with permission.
Figure 9
Figure 9
Immunofluorescence of FLAG-SK1a channels and folate chemoreceptor (FBP) a GPI anchored protein with antibodies against the FBP. Note that both are in the cilia, while FBP is in both the cilia and cell membrane. Only the merged images are shown here, but all controls are available [10]. FLAG control is a cell microinjected with FLAG-pPXV vector and fed RNAi empty vector bacteria. FLAG-SK1a control is a cell expressing FLAG-SK1a and fed bacteria with an RNAi empty vector. “BBS8”cells are expressing FLAG-Sk1a channel and are also depleted of BBS8 RNA by RNAi. Cells were immune stained with anti-FLAG (red) and anti-FBP (green) antibodies. These images are from a larger published study with more BBS genes silenced, and with similar results. Note that Sk1a is missing from cilia after silencing BBS8, but the FBP remains in the cilia. Reproduced from [8], with permission.
Figure 10
Figure 10
Ultrastructure of the Paramecium transition zone (A) cross-section of a Paramecium transition zone showing the terminal plate (TP), intermediate plate (IP), and axosomal plate along with the axosome (A) and loosely packed ring (LR). (B) The 3D reconstruction of the Paramecium transition zone and beginning of the cilium that is attached. The ciliary membrane (A) is contiguous with the plasma membrane (O). the alveolar sacs that lay just below the outer (P) and inner (Q) membrane. Extending above the transition zone are the central tubules (C), only one of which enters the axosome (H). The peripheral doublets (B) of the cilium also begin above the plaque particles (D) that cover the plaque complex (E). There is loosely packed ring material (G) that surround the axosome that site ajust above the axosomal curved plate (I). The ciliary necklace (F) surrounds the cilium near the rings that connect the peripheral tubes (J). The intermediate plate (K) sits at the center just above the terminal plate (L). Transitional fibers (M) and projections from the peripheral tubules (N) are also shown. Reproduced from [86], with permission.
Figure 11
Figure 11
Localization of TZ proteins in Paramecium. (A) Paramecia labeled by 1D5 to identify the basal bodies showing the invariant field (double basal body units) at the anterior of the cell and the mixed field (both single- and double-basal body units) (A’). The antibody also decorates the cilia (A’’), scale bars are labeled. Electron microscopy images show longitudinal sections of these basal bodies in the invariant (left) and mixed (right) fields. (B) The yellow arrows show the terminal plate (bottom), the intermediate plate (middle) and the axosomal plate (top) present in the TZ (pink vertical double-arrows). Note that the basal body on the far right from the invariant field shows a shorter, more compressed TZ, as this basal body does not have a cilium, scale bars are 200 nm. (C) Graphical representation of the mean length of the TZ in ciliated and non-ciliated basal bodies (56 ciliated and 100 non-ciliated basal bodies were measured, **** p < 0.001, unpaired 2-tailed t-test. (D) Paramecium expressing one of the five different TZ proteins (TMEM107, TMEM216, CEP290, RPGRIP1L, and NPHP4) tagged with GFP. Basal bodies are also stained (pink, 1D5), invariant zone is highlighted in white on the shown TMEM216-GFP expressing cell. Notice that the GFP-tagged protein is only shown at the distal part of ciliated basal bodies (green staining). Note that NPHP4-GFP can also be seen at the proximal end of the basal body. (E) STED images showing the 9-fold symmetrical localization of each protein and the diameter of these rings were measured to produce a graph (F) of the mean diameter with standard deviation of these proteins with the top-right showing respect to the basal body and to the ciliary membrane. (G) shows a representative TEM image of the localization of the different GFP-tagged proteins. The lower panels are transverse sections of basal bodies at the plane of the axosomal plate. The upper-right panels show the position of gold beads using diagrams. The left panels all show longitudinal views. Although the proteins all have different diameters, they are all found at the axosomal plate (black arrowhead). Image from [11], reproduced with permission.
Figure 12
Figure 12
Chaotic orientation of the striated rootlet of MKS3-depleted cells. Control cells (A) and MKS3-depleted cells (B) were stained with anti-Glu-α-tubulin (red; basal bodies) and anti-striated rootlets (SRs) (also called anti-kinetodesmal fibers; green). Yellow arrows in (A) and (B) indicate the contractile vacuoles on the dorsal surface of these cells. SRs project from the basal bodies. In two basal body units, SRs project only from the posterior basal body (a; dotted yellow arrows). SRs project toward the anterior of the cell in a highly organized manner along the basal body row (kinety). Cells depleted of MKS3 show chaotic organization of the SRs, which project in every direction (b and b’). Scale bar: 10 µm. Reproduced from [12], with permission.
Figure 13
Figure 13
Depletion of MKS5 cause loss of cilia but does not affect the straight basal body rows and Table 1. D5 and anti-SR antibody to visualize the basal body units (green) and SRs (red) respectively on the cell surface. Panels A and B show the basal body row alignment and SRs organization in the control cell. In the control cell basal body rows remain straight and SRs show a highly ordered organization. SR emanates from the basal body unit and extends towards the anterior pole of the cell. Panels C and D show the basal body row alignment and SRs in the MKS5 depleted cells. The phenotype of the cell is similar to the control cell. Panels E and F show the basal body row alignment and SRs in the MKS3 depleted cell. In the MKS3 depleted cell, basal body rows are misaligned and SRs have a disordered organization on the cell surface. Scale bars are 15 µm in (A,C,E) and 3 µm in (B,D,F). Reproduced from [13] with permission.
Figure 14
Figure 14
Depletion of MKS5 affects the localization of B9D2 protein in TZ of the basal body. All cells were treated with anti-Tetrahymena centrin and anti-GFP antibody to visualize the basal body units (red) and GFP-B9D2 protein in the cell. In all the images, the yellow box highlighted area is enlarged to show the basal body rows and GFP localization in the GFP-B9D2 expressing cell. Panels A and B show the basal body row alignment and GFP-B9D2 localization in the control cell. In the control cell, basal body (red) rows remain straight with GFP-B9D2 protein localization at the TZ of the basal body in a single basal body unit. In two basal body units, only the posterior basal body has the signal for GFP-B9D2 protein. Panels C and D show the basal body row alignment and GFP-B9D2 protein localization in the MKS5 depleted cell. In the MKS5 depleted cell, basal body rows remain straight like the control cell but GFP-B9D2 localization shows a remarkable difference compared to the control cell. Both the single basal body and the posterior basal body of the two basal body unit lack the localization of B9D2 protein in the MKS5 depleted cell. Panels E and F show the basal body row misalignment and localization of B9D2 protein in the MKS3 depleted cell. In the MKS3 depleted cells, cell basal body rows are misaligned and GFP-B9D2 protein shows very dispersed and diffused localization. Scale bars are 10 µm in (A,C,E) and 3 µm in (B,D,F). Reproduced with permission from [13].
Figure 15
Figure 15
Striated Rootlet Disruption Control (A,C) and Structural Group 1 Depleted Cells (B,D). (A,C) Basal body row alignment and SR appearance in control cells (A) or cells with SR Structural Group 2 depleted by RNAi. Basal bodies are green (1D5 antibody) and SRs are red (anti-SR antibody). Note that in the Structural Group 2 SR RNAi treated cells, SRs are shorter and pointing in directions out of alignment with the basal body rows as in the Control Cell (A). (B,D) Swimming patterns of cells taken by darkfield microscopy. Shown here are Control (B) and Structural Group-1 depleted (by RNAi) cells (D). Scale bar is 1 mm. Reproduced from [90], with permission.
Figure 16
Figure 16
A camera lucida drawing shows the ventral surface of Paramecium. The anterior two basal body field (2-bb field), or invariant zone, contains all two basal body units, each with a cilium arising. The mixed field wraps around the cell and contains cortical units with either a single- or double-basal body. In this region, only the posterior of the basal bodies has a cilium arising from it. The single basal body field (1-bb field) at the posterior of the cell contains cortical units with only one basal body, each with a cilium arising (Image from [96], reviewed in [98]). Reproduced with permission.
Figure 17
Figure 17
Striated rootlet structure and alignment disrupted by RNAi. Depletion of SR proteins leaves the angles between the rootlets unaffected. Images show staining of all three rootlets (TR, green; PR green; SR, red). TR and PR and stain green with anti-acetylated tubulin. Basal body stains green with 1D5. SR is stained red with anti-SR antibodies (courtesy of J. Beisson). TR stretches across the basal body row; PR stretches toward the posterior; SR points toward the anterior, in alignment with the basal body row. Scale bars 3 µm. White circles denote the position of basal bodies with all three rootlets visible that were used in measuring the angles between rootlets. At least 100 basal bodies with rootlets were examined for each condition. Pick a white circle in the first two panels and see that there clearly are two green rootlets stretching out from the basal body and a red rootlet in line with the row of basal bodies. The control image shows the three rootlets attached to the basal body, with the SRs in red joining to form a clear line pointing toward the anterior of the cell. No change was expected for Paralog Group 2 depleted cells, but Structural Group depleted cells were expected to show misalignments, as they clearly do here. When the angles between the three rootlets were measured, there was no change in the angles despite what looks like a rotation of the basal body and rootlets out of alignment with their row. Reproduced from [90], with permission.
Figure 17
Figure 17
Striated rootlet structure and alignment disrupted by RNAi. Depletion of SR proteins leaves the angles between the rootlets unaffected. Images show staining of all three rootlets (TR, green; PR green; SR, red). TR and PR and stain green with anti-acetylated tubulin. Basal body stains green with 1D5. SR is stained red with anti-SR antibodies (courtesy of J. Beisson). TR stretches across the basal body row; PR stretches toward the posterior; SR points toward the anterior, in alignment with the basal body row. Scale bars 3 µm. White circles denote the position of basal bodies with all three rootlets visible that were used in measuring the angles between rootlets. At least 100 basal bodies with rootlets were examined for each condition. Pick a white circle in the first two panels and see that there clearly are two green rootlets stretching out from the basal body and a red rootlet in line with the row of basal bodies. The control image shows the three rootlets attached to the basal body, with the SRs in red joining to form a clear line pointing toward the anterior of the cell. No change was expected for Paralog Group 2 depleted cells, but Structural Group depleted cells were expected to show misalignments, as they clearly do here. When the angles between the three rootlets were measured, there was no change in the angles despite what looks like a rotation of the basal body and rootlets out of alignment with their row. Reproduced from [90], with permission.
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