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Random cross sections through the distal parts of basal bodies from cytoskeletons fixed and embedded for standard electron microscopy. Scale bar: 0.1 m. Basal body doublets/triplets 2, 4, 7, and 9 are numbered, and some transitional fibers (tf) are marked. The left section is slightly more distal than the right, but both show the “acorn” in the lower left part of the basal body lumen attached to doublets/triplets 7, 8, 9, 1, and 2. The V-shaped filament system occupies the area opposite the acorn in the more distal section (arrows in panel 37), whereas, in the more proximal section (38), it extends into the area of the acorn (actually slightly proximal to the acorn; see also Fig. 5, panels 39 to 44), radiating into several thin filaments which link to doublets/triplets 8, 9, and 1. For a schematic representation of the acorn and V-shaped filament system seen in panels 37 and 38, see Fig. 5, panels 41 and 42, respectively. To be consistent with the other schematic drawings, the basal bodies have been rotated counterclockwise by 90° compared to the basal bodies shown in panels 37 and 38.
Source publication
In the flagellate green alga Chlamydomonas reinhardtii the Ca2+-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct
structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional
region, and the axoneme. To ex...
Contexts in source publication
Context 1
... 37, and 5, panel 41). Additional thinner filaments connect the thick filaments also to triplets/doublets 3 and 6 ( Fig. 4, panel 37, and 5, panel 41). In longitudinal sections through basal bodies the two V-shaped filaments are oriented almost perpendicular to the long axis of the basal body and in the center of the basal body lumen they are linked to the fine filament emanating from the flagellar transitional region (Fig. 1, panels 10, 11, and 20, and 5, panel 39). From the junction with the fine filament, the V-shaped filament system continues to extend further downwards (i.e., proximally), forming an angle of about 35° to the transverse basal body axis. The fila- ment system traverses the lumen of the basal body, reaching the opposite surface almost 200 nm from its junction with the fine filament (Fig. 1, panels 10, 11, and 20, and 5, panel 39). At this basal body surface, the V-shaped filament system radiates into usually four separate fibers, which contact basal body triplets 8, 9, and 1 and the interlinkers to triplets 2 and 7 (Fig. 4, panel 38, and 5, panel 42). The V-shaped filament system contains centrin (Fig. 1, panels 7, 19, and 20, and 2, panels 21 to 23). Due to the oblique orientation of the V-shaped filament system (see above) centrin displays a rotationally asymmetric distribution in the distal part of the basal body. At the level of the acorn, centrin is predominantly located in the part of the basal body lumen not containing the acorn (Fig. 2, panel 21, and 5, panel 41), whereas at a slightly lower (proximal) level, centrin is associated mostly with the opposite part of the basal body lumen underlying the acorn (Fig. 1, panel 20, 2, panel 22, and 5, panels 39 and 42). The asymmetric distribution of centrin is also evident in cross sections through the distal part of the basal body (Fig. 1, panel 13, and 2, panels 25 and 26). Another novel localization of centrin in the basal apparatus is that of the transitional fibers (Fig. 1, panels 13, 14, and 17, 2, panels 25 to 27, and 5, panels 41 to 43). The nine fibers project radially and in counterclockwise orientation from each basal body triplet and in intact cells attach the basal bodies to the plasma membrane. They are associated with the triplets over a considerable length of the distal part of the basal bodies (Fig. 2, panel 23, and 5, panel 39). Since centrin labeling of the transitional fibers was not intensive, a statistical analysis of centrin distribution was performed (Table 1). In immune- labeled cross sections through basal bodies ( n ϭ 54) about 87% of the basal bodies revealed labeling of at least one transitional fiber (with an average labeling density of 1.8 gold particles/ transitional fiber; on average 2.9 Ϯ 1.3 [standard deviation] transitional fibers were labeled per basal body). In preimmune controls ( n ϭ 34) only 6% of the basal bodies displayed gold particles over transitional fibers (average labeling density: 1 gold particle/transitional fiber). Labeling was more prominent in the broader, proximal parts of the transitional fibers compared to the thinner distal parts (Fig. 1, panels 13, 14, and 17, and 2, panels 26 and 27) and also extended into the linkers interconnecting basal body triplets (Fig. 1, panels 14 and 17, and 2, panel 27). Of the remaining centrin localizations in the basal appara- tus, the two most prominent were the distal connecting fiber and the nucleus-basal body connectors, two well-known centrin-containing structures. In our preparations, the most heavily labeled basal apparatus component was the distal connecting fiber (Fig. 1, panels 10, 15, 16, 19, and 20, 2, panel 21, and 3, panels 31, 33, and 35). The distal connecting fiber is cross-striated and interconnects the two flagellum-bearing basal bodies (Fig. 1, panel 11, 2, panel 29, and 5, panel 39). Although there was some indication that centrin labeling was more prominent over the electron-dense striations compared to the more lightly staining regions of the fiber (Fig. 1, panels 10 and 20, and 2, panel 21), we did not investigate this in a quantitative manner. Of greater interest in the present context was the observation that centrin labeling in the distal connect- ing fiber apparently extends into the basal body lumen and is continuous with the downward-bending part of the V-shaped filament system. This is most clearly documented in longitudinal sections through basal bodies (Fig. 1, panels 19 and 20, and 2, panel 22); but that centrin labeling from the distal connecting fiber extends at least into the linkers between basal body triplets 9, 1, and 2 was also observed in cross sections through basal bodies (Fig. 2, panel 28). Serial cross sections, however, due to the oblique orientation of the V-shaped filament system in the basal body lumen (see above), cannot reveal the continuity between the distal connecting fiber and the V-shaped filament system (Fig. 1, panels 13 to 15). The two NBBCs extend from the flagellum-bearing basal bodies and connect the basal apparatus with the nucleus. ...
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... 37, and 5, panel 41). Additional thinner filaments connect the thick filaments also to triplets/doublets 3 and 6 ( Fig. 4, panel 37, and 5, panel 41). In longitudinal sections through basal bodies the two V-shaped filaments are oriented almost perpendicular to the long axis of the basal body and in the center of the basal body lumen they are linked to the fine filament emanating from the flagellar transitional region (Fig. 1, panels 10, 11, and 20, and 5, panel 39). From the junction with the fine filament, the V-shaped filament system continues to extend further downwards (i.e., proximally), forming an angle of about 35° to the transverse basal body axis. The fila- ment system traverses the lumen of the basal body, reaching the opposite surface almost 200 nm from its junction with the fine filament (Fig. 1, panels 10, 11, and 20, and 5, panel 39). At this basal body surface, the V-shaped filament system radiates into usually four separate fibers, which contact basal body triplets 8, 9, and 1 and the interlinkers to triplets 2 and 7 (Fig. 4, panel 38, and 5, panel 42). The V-shaped filament system contains centrin (Fig. 1, panels 7, 19, and 20, and 2, panels 21 to 23). Due to the oblique orientation of the V-shaped filament system (see above) centrin displays a rotationally asymmetric distribution in the distal part of the basal body. At the level of the acorn, centrin is predominantly located in the part of the basal body lumen not containing the acorn (Fig. 2, panel 21, and 5, panel 41), whereas at a slightly lower (proximal) level, centrin is associated mostly with the opposite part of the basal body lumen underlying the acorn (Fig. 1, panel 20, 2, panel 22, and 5, panels 39 and 42). The asymmetric distribution of centrin is also evident in cross sections through the distal part of the basal body (Fig. 1, panel 13, and 2, panels 25 and 26). Another novel localization of centrin in the basal apparatus is that of the transitional fibers (Fig. 1, panels 13, 14, and 17, 2, panels 25 to 27, and 5, panels 41 to 43). The nine fibers project radially and in counterclockwise orientation from each basal body triplet and in intact cells attach the basal bodies to the plasma membrane. They are associated with the triplets over a considerable length of the distal part of the basal bodies (Fig. 2, panel 23, and 5, panel 39). Since centrin labeling of the transitional fibers was not intensive, a statistical analysis of centrin distribution was performed (Table 1). In immune- labeled cross sections through basal bodies ( n ϭ 54) about 87% of the basal bodies revealed labeling of at least one transitional fiber (with an average labeling density of 1.8 gold particles/ transitional fiber; on average 2.9 Ϯ 1.3 [standard deviation] transitional fibers were labeled per basal body). In preimmune controls ( n ϭ 34) only 6% of the basal bodies displayed gold particles over transitional fibers (average labeling density: 1 gold particle/transitional fiber). Labeling was more prominent in the broader, proximal parts of the transitional fibers compared to the thinner distal parts (Fig. 1, panels 13, 14, and 17, and 2, panels 26 and 27) and also extended into the linkers interconnecting basal body triplets (Fig. 1, panels 14 and 17, and 2, panel 27). Of the remaining centrin localizations in the basal appara- tus, the two most prominent were the distal connecting fiber and the nucleus-basal body connectors, two well-known centrin-containing structures. In our preparations, the most heavily labeled basal apparatus component was the distal connecting fiber (Fig. 1, panels 10, 15, 16, 19, and 20, 2, panel 21, and 3, panels 31, 33, and 35). The distal connecting fiber is cross-striated and interconnects the two flagellum-bearing basal bodies (Fig. 1, panel 11, 2, panel 29, and 5, panel 39). Although there was some indication that centrin labeling was more prominent over the electron-dense striations compared to the more lightly staining regions of the fiber (Fig. 1, panels 10 and 20, and 2, panel 21), we did not investigate this in a quantitative manner. Of greater interest in the present context was the observation that centrin labeling in the distal connect- ing fiber apparently extends into the basal body lumen and is continuous with the downward-bending part of the V-shaped filament system. This is most clearly documented in longitudinal sections through basal bodies (Fig. 1, panels 19 and 20, and 2, panel 22); but that centrin labeling from the distal connecting fiber extends at least into the linkers between basal body triplets 9, 1, and 2 was also observed in cross sections through basal bodies (Fig. 2, panel 28). Serial cross sections, however, due to the oblique orientation of the V-shaped filament system in the basal body lumen (see above), cannot reveal the continuity between the distal connecting fiber and the V-shaped filament system (Fig. 1, panels 13 to 15). The two NBBCs extend from the flagellum-bearing basal bodies and connect the basal apparatus with the nucleus. ...
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... reinhardtii cytoskeletons contains filaments, some of which are attached to the A-tubule feet at the inner surface of the triplets (13). The quantity of these filaments varied among basal bodies, suggesting differential extraction of filaments during isolation or preparation of cytoskeletons for electron microscopy. Thus, no attempt was made to quantify the labeling or analyze its distribution. We note, however, that the basal body lumen reveals centrin labeling in the area where the NBBC is linked to triplets 7 and 8 (Fig. 1, panel 16, and 2, panel 29) and that a peculiar centrin localization was observed on several occasions at the proximal end of a basal body near the central hub of the cartwheel from which a fine filament extends towards the proximal end of the opposite basal body (Fig. 3, panels 35 and 36). In addition to the flagellum-bearing basal bodies, most basal apparatuses of isolated cytoskeletons also contained two probasal bodies (Fig. 2, panels 29 and 30, 3, panel 34, and 5, panels 39 and 44 [schematic presentation]). Probasal bodies are very short (about 100 nm in length) (13) and consist of a microtubular cylinder of nine triplets and an elaborate cartwheel which fills the lumen of the probasal body almost completely and protrudes proximally from the microtubular triplets in a coni- cal shape (Fig. 3, panel 31). The distal end of a probasal body contains an acorn (13) (Fig. 5, panels 39 and 44). Here we show that centrin is present in the probasal bodies (Fig. 2, panel 30, and 3, panels 31 to 33). In immune-labeled cross sections through probasal bodies ( n ϭ 93) 61% of the probasal bodies displayed gold particles (with an average labeling density of 3.5 gold particles/probasal body; Table 1). In preimmune controls only 7% of the probasal bodies ( n ϭ 115) were labeled with gold particles (average labeling density: 1.2 gold particles/probasal body). Centrin labeling was exclusively confined to the distal end of the probasal bodies, as shown in longitudinal sections through probasal bodies (Fig. 2, panel 30, and 3, panels 31 and 32). In cross sections through probasal bodies ( n ϭ 59) centrin labeling was rotationally asymmetric, with twice as many probasal bodies (54%) labeled in the luminal part opposite the acorn compared to the luminal part containing the acorn (24%; label in the remaining probasal bodies was distributed equally over both luminal parts). We have previously shown that probasal bodies contain the typical V-shaped filament system near their distal end (13) (see also schematic presentations in Fig. 5, panels 39 and 44). We also searched for a centrin connective between probasal bodies and the flagellum-bearing basal bodies, but without success. However, we consistently observed a centrin “patch” on the two-stranded microtubular flagellar root near the site where the probasal body is attached to the two-stranded root (Fig. 2, panel 29, 3, panels 33 and 34, and 5, panel 44 [schematic presentation]). The various centrin localizations in the basal apparatus of isolated cytoskeletons of Chlamydomonas reinhardtii documented in this study by postembedding serial section immunoelectron microscopy are summarized in the schematic presentations of Fig. 5, panels 39 to 44. Our interpretation of the distribution of centrin in the basal apparatus of Chlamydomonas reinhardtii is that centrin is intimately associated with a complex filamentous network which pervades the basal apparatus and extends from the nuclear surface to the proximal end of the flagella and is here termed the centrin scaffold. The distribution of centrin in the basal apparatus of the flagellate green alga Chlamydomonas reinhardtii has been studied in detail by serial section postembedding immunoelectron microscopy of cytoskeletons isolated from interphase cells. Although centrin was first discovered in a flagellate green alga (60) and its localization in the basal apparatus of algae has been well investigated at both the light microscopy and electron microscopy levels (reviewed in reference 44), several novel centrin localizations were discovered during the course of this study. Since centrin was localized in isolated cytoskeletons, the likelihood of an artificial association of centrin with basal apparatus components during cell disruption should be considered. We have previously shown that the basal apparatus of isolated cytoskeletons of C. reinhardtii remains structurally intact and that its detailed ultrastructure corresponds to that of the in situ basal apparatus (13). Several functions of the basal apparatus have been reconstituted in isolated cytoskeletons such as the contraction of the centrin-containing NBBCs (77), and we have confirmed all previously known in situ localizations of centrin in C. reinhardtii (61) also in the isolated cytoskeletons. Searching published micrographs for the novel centrin localizations in C. reinhardtii yielded a positive hit for the distal part of the basal body lumen (Fig. 4 in reference 61 and 6A in reference 63 [both in situ localizations]). That the distal parts of basal bodies/centrioles contain centrin was first described in brown algal gametes (24), but later also documented from amoeboflagellates to humans (37, 49). It is likely that centrin is located in the distal lumen of the basal body/ centriole in most if not all eukaryotes. The presence of centrin in novel localizations in the Chlamydomonas basal apparatus is thus most likely not caused by the attraction of soluble centrin to basal apparatus components during cell disruption, but is genuine. However, the possibility of an artificial redistribution of centrin during cell disruption could be excluded by an in- vestigation of cryofixed whole cells, which would also allow the localization of possible pools of soluble centrin within the cell/in the vicinity of the basal apparatus. The failure to recognize centrin as a component of the basal body proper in Chlamydomonas (see, e.g., reference 28) is likely related to the preponderance of the preembedding technique (which precludes access of gold particles to the basal body lumen) and the difficulty to distinguish false positives from true centrin labeling when labeling is weak and fixation/ contrast suboptimal as in many previous postembedding studies. An additional advantage of using isolated cytoskeletons is that the high density of basal apparatuses supports a statistical approach which significantly increases the confidence level of even weak labeling densities. When isolated cytoskeletons of C. reinhardtii were recently used to establish a high-resolution structural map of the interphase basal apparatus, it was antic- ipated that the data would “facilitate the precise mapping of previously identified basal apparatus proteins” (13). This ex- pectation was confirmed upon probing centrin localization in isolated cytoskeletons: in the distal part of the basal body, centrin was found to be associated with a specific rotationally asymmetric structure, the V-shaped filament system, which had been discovered during the previous study. Interestingly, both the V-shaped filament system and centrin are already present in the “dormant” probasal bodies of C. reinhardtii (reference 13 and this study), suggesting that the V-shaped filament system is the essential centrin-containing structure of a developing basal body. In various other systems, centrin has been found to be associated with developing (nascent) basal bodies/ centrioles (reviewed in reference 5), e.g., in the blepharoplast of Marsilea (26), in fibrogranular precursors of centrioles of human epithelial cells (29, 30), and at the surface of fibrous probasal body precursors in flagellate green algae (31). In addition, functional/genetic evidence has been accumulating that centrin is involved in basal body assembly/duplication (28, 46, 49, 62). It is thus possible that the V-shaped filament system or its precursor in conjunction with other proteins is involved in probasal body formation/assembly. Centrin has also been discovered in the transitional fibers. The transitional fibers have been inferred to function as docking sites for intraflagellar transport particles and their motors destined for the flagella (9). Intraflagellar transport (IFT) is required for the assembly and maintenance of flagella and involves the transport of non-membrane-bound macromolec- ular protein complexes (IFT particles) from the cell body to the tip of the flagellum and back (reviewed in references 56 and 66). Interestingly, centrin is known as a light chain associated with one type of inner dynein arm in axonemes of C. reinhardtii (33, 52) and has been implicated in different ciliary functions (14, 15). Here, we have shown specific centrin labeling of outer axonemal doublets consistent with the biochemical evidence. However, the centrin labeling density of both the axonemes and the transitional fibers was relatively low (see Results). Loss of centrin during isolation of the cytoskeletons and/or preparatory steps for electron microscopy may be one explanation for the observed weak labeling. In isolated mam- malian centrosomes, centrin is only extracted when drastic conditions such as 8 M urea or high temperatures (45°C) are applied (49). One of the inner dynein arm complexes (2B) containing centrin, however, was shown to be extracted by low concentrations of the detergent Nonidet P-40 (33). It is thus conceivable that, depending on whether centrin is associated with (contractile) filaments or is part of a heavy-chain dynein complex, it may be differentially stable to extraction. The weak centrin labeling of the transitional fibers may be related to extraction of cargo centrin in transit to the IFT machinery (the latter appears to be concentrated at the distal ends of the transitional fibers [9]). There is strong evidence that inner dynein arm subunits are transported in and out of the flagellum by IFT. Inactivation of the ...
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... (also known as caltractin) is a member of the highly conserved superfamily of calcium-binding EF-hand proteins and occurs in all eukaryotic cells, being mostly associated with centrosomes (58, 64). It was first discovered in flagellate green algae, where it is located in the basal apparatus (the part of the cytoskeleton anchoring the flagella) and involved in the contraction of calcium-sensitive filaments (42, 60) which, in general, play an important role in the dynamic behavior of centrosomes by controlling the position and orientation of centrosomal structures (58, 64). Analysis of centrin mutants and RNA interference strains deficient in centrin in the green algal model system Chlamydomonas reinhardtii suggested that centrin is essential for basal body duplication and segregation during cell division (28, 39, 73), functions also deduced for centrin homologues in vertebrates (46, 62) and yeast spp. (3, 50, 69). More recently, novel centrin functions have been discovered such as involvement in recombinational DNA repair through modulation of the nucleotide excision repair pathway in humans and plants (2, 47), a role in the nuclear mRNA export machinery in yeast (11), regulation of calcium-depen- dent ciliary activities in ciliates (14, 15), and signal transduction in vertebrate photoreceptors (53). How centrin exerts these different functions at the molecular level is, however, still unknown although considerable progress has recently been made through in vitro studies of centrin and its interaction with target peptides (8, 19, 20, 40, 75). In some organisms, the functional diversity of centrin may be related to the presence of different centrin isotypes, e.g., four centrin proteins (centrin1p to centrin4p), which are differentially expressed in different tissues and cell types, have been identified so far in mammals (10, 12, 29, 34, 35, 45). In the ciliate Paramecium , three centrin genes have been character- ized to date (38) and in another ciliate ( Paraurostyla ) evidence has been presented for differential localization of two centrin isotypes (36). In Chlamydomonas reinhardtii and other flagellate green algae, in which centrin localization and function have been well studied for over 20 years, only a single centrin gene is present (6, 21). The protein, however, has been found at six distinct locations within the cytoskeleton: the nucleus-basal body connector (NBBC), the distal connecting fiber, the flagellar transitional region, the basal body lumen, the axoneme, and the probasal bodies (22, 31, 42, 60, 63, 67, 77). It has thus been suggested that the specificity of centrin localization in green algae may perhaps be provided by different centrin-binding proteins (27). Centrin-binding proteins have been difficult to identify and are mostly known only from yeast (i.e., Kar1p, Kic1p, Mps3p, and Sfi1p) (7, 23, 25, 70, 72). One of these proteins, Sfi1p, however, is also present in mammals (25) and is apparently homologous to a centrin-binding protein (CBP1.1) previously identified in the flagellate green alga Spermatozopsis similis (71). This protein contains multiple internal repeats, with each repeat binding one centrin molecule. It is likely that Sfi1p forms filaments on which centrin molecules are arranged like a string of pearls, providing an elegant mechanism for filament contraction by supercoiling (25, 57). Here we show using serial section postembedding immunoelectron microscopy of isolated cytoskeletons of Chlamydomonas reinhardtii that the previously identified distinct localizations of centrin are part of a continuous filamentous scaffold that extends from the nucleus to the base of the axonemes. We describe the intricate association of the centrin scaffold with the basal bodies and demonstrate rotational asymmetry of centrin distribution in the distal lumen of the basal bodies. The distal ends of basal bodies are thought to represent a center of rotational asymmetry in the cytoskeleton of flagellate/ciliate cells (13). The ultrastructure of the Chlamydomonas reinhardtii basal apparatus has been recently reevaluated in detail using dual- axis electron tomography of whole cells (48) and serial thin- section electron microscopy of isolated cytoskeletons (13), and the reader is referred to these publications for details of ter- minology and structural components of the basal apparatus. In this communication we used serial section postembedding immunogold electron microscopy on isolated cytoskeletons of C. reinhardtii to localize centrin in the basal apparatus. The results are illustrated in Fig. 1 to 4 and summarized in the schematic presentation of Fig. 5. In addition, we performed extensive statistical analyses of the distribution of gold particles (coated with secondary antibody) over individual basal apparatus components by comparing sections exposed to immune versus preimmune centrin IgGs (Table 1). In general, postembedding immunogold electron microscopy has the advantage over the preembedding technique that antigens inaccessible to gold particles (i.e., those located within other structural components such as basal bodies) become exposed in sections. Progress in preserving fine details of non- osmicated cellular structures and the establishment of a detailed structural map of all basal apparatus components of C. reinhardtii (13) facilitate assignment of gold particles (within the resolution determined by the size of the gold-antibody complexes) to specific structures, thus eliminating one of the traditional problems of the postembedding technique. Our preimmune controls revealed no or extremely few gold particles (Fig. 1, panels 11 and 18; Table 1), thus increasing the specificity and sensitivity of the labeling. As a critical test of this approach we investigated centrin labeling over axonemal cross sections (Table 1; Fig. 1, panels 1 to 3). About 42% of the axonemes were labeled (with an average labeling density of 1.8 gold particles/axonemal cross section; n ϭ 536). In preimmune controls, only 6% of the axonemes were labeled ( n ϭ 334). Next, we analyzed where this labeling occurred, probing 117 labeled, randomly chosen axonemal cross sections (displaying a total of 194 gold particles). We found that 96 (82%) of the cross sections showed labeling over the outer doublet microtubules and 21 (18%) were labeled in other axonemal areas (mostly over the central pair microtubules). Thus, centrin labeling in the axoneme was nonrandom and mostly confined to the outer doublet microtubules (often associated with the A tubule; Fig. 1, panels 1 to 3). No centrin labeling of the outer doublet microtubules was observed in the transitional region (the most proximal region) of the axoneme. Centrin is known as a light chain associated with one type of inner dynein arm (33, 52) (see Discussion), which is not present in this region (18). In contrast to the relatively weak anticentrin labeling in the axoneme, the star-like pattern and ring-like hub of the flagellar transitional region (well-known locations of centrin) were strongly labeled by gold particles (Fig. 1, panels 4 to 10 and 12). The statistical analysis revealed that 85% of the cross sections analyzed ( n ϭ 39) were labeled, with an average labeling density of 9.9 gold particles/transitional region (Table 1). In the preimmune control, only 3% of the transitional regions were labeled and the labeling density was low (on average 1 gold particle/transitional region). Because of the high labeling density, the distribution of label over the transitional region was not analyzed statistically. However, we noticed that the labeling was more intense over the central ring-like hub compared to the peripheral star-like pattern (Fig. 1, panels 4 to 10). In longitudinal sections of the transitional region both the distal and proximal stellate structures were labeled (Fig. 1, panels 7 to 10). Interestingly, in such sections we often encountered string-like centrin labeling extending centrally from the distal end of the distal stellate structure through the proximal stellate structure to the distal end of the basal body (Fig. 1, panels 7 to 9). The latter part of the string-like labeling corresponds to a previously identified fine filament that extends from the proximal stellate structure to a V-shaped filament system at the distal end of the basal body (13) (compare also Fig. 1, panels 8 and 9 with panels 10 and 11). The distal end of the basal body has been previously shown to contain two different filament systems, both positioned in a rotationally asymmetric manner, namely, the “acorn” and the V-shaped filament system (13) (Fig. 4, panels 37 and 38, and 5, panels 41 and 42 [schematic presentations]). The acorn, a fiber outlining the shape of an acorn in cross sections through basal bodies, is attached to triplets 7, 8, 9, 1, and 2 and is thus located in the part of the basal body facing the opposite basal body (Fig. 5, panels 39, 41, and 44). In longitudinal sections through basal bodies the V- shaped filament system is obliquely oriented (Fig. 1, panels 10, 11, and 20, and 5, panel 39). Its upper (distal) part is located at nearly the same level but opposite to the acorn and consists mainly of two 8-nm-thick filaments, which extend from microtubule triplets/doublets 4 and 5 (mostly the A tubules) into the center of the basal body lumen, where they converge, hence their V-shape in cross sections through basal bodies (Fig. ...
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... (also known as caltractin) is a member of the highly conserved superfamily of calcium-binding EF-hand proteins and occurs in all eukaryotic cells, being mostly associated with centrosomes (58, 64). It was first discovered in flagellate green algae, where it is located in the basal apparatus (the part of the cytoskeleton anchoring the flagella) and involved in the contraction of calcium-sensitive filaments (42, 60) which, in general, play an important role in the dynamic behavior of centrosomes by controlling the position and orientation of centrosomal structures (58, 64). Analysis of centrin mutants and RNA interference strains deficient in centrin in the green algal model system Chlamydomonas reinhardtii suggested that centrin is essential for basal body duplication and segregation during cell division (28, 39, 73), functions also deduced for centrin homologues in vertebrates (46, 62) and yeast spp. (3, 50, 69). More recently, novel centrin functions have been discovered such as involvement in recombinational DNA repair through modulation of the nucleotide excision repair pathway in humans and plants (2, 47), a role in the nuclear mRNA export machinery in yeast (11), regulation of calcium-depen- dent ciliary activities in ciliates (14, 15), and signal transduction in vertebrate photoreceptors (53). How centrin exerts these different functions at the molecular level is, however, still unknown although considerable progress has recently been made through in vitro studies of centrin and its interaction with target peptides (8, 19, 20, 40, 75). In some organisms, the functional diversity of centrin may be related to the presence of different centrin isotypes, e.g., four centrin proteins (centrin1p to centrin4p), which are differentially expressed in different tissues and cell types, have been identified so far in mammals (10, 12, 29, 34, 35, 45). In the ciliate Paramecium , three centrin genes have been character- ized to date (38) and in another ciliate ( Paraurostyla ) evidence has been presented for differential localization of two centrin isotypes (36). In Chlamydomonas reinhardtii and other flagellate green algae, in which centrin localization and function have been well studied for over 20 years, only a single centrin gene is present (6, 21). The protein, however, has been found at six distinct locations within the cytoskeleton: the nucleus-basal body connector (NBBC), the distal connecting fiber, the flagellar transitional region, the basal body lumen, the axoneme, and the probasal bodies (22, 31, 42, 60, 63, 67, 77). It has thus been suggested that the specificity of centrin localization in green algae may perhaps be provided by different centrin-binding proteins (27). Centrin-binding proteins have been difficult to identify and are mostly known only from yeast (i.e., Kar1p, Kic1p, Mps3p, and Sfi1p) (7, 23, 25, 70, 72). One of these proteins, Sfi1p, however, is also present in mammals (25) and is apparently homologous to a centrin-binding protein (CBP1.1) previously identified in the flagellate green alga Spermatozopsis similis (71). This protein contains multiple internal repeats, with each repeat binding one centrin molecule. It is likely that Sfi1p forms filaments on which centrin molecules are arranged like a string of pearls, providing an elegant mechanism for filament contraction by supercoiling (25, 57). Here we show using serial section postembedding immunoelectron microscopy of isolated cytoskeletons of Chlamydomonas reinhardtii that the previously identified distinct localizations of centrin are part of a continuous filamentous scaffold that extends from the nucleus to the base of the axonemes. We describe the intricate association of the centrin scaffold with the basal bodies and demonstrate rotational asymmetry of centrin distribution in the distal lumen of the basal bodies. The distal ends of basal bodies are thought to represent a center of rotational asymmetry in the cytoskeleton of flagellate/ciliate cells (13). The ultrastructure of the Chlamydomonas reinhardtii basal apparatus has been recently reevaluated in detail using dual- axis electron tomography of whole cells (48) and serial thin- section electron microscopy of isolated cytoskeletons (13), and the reader is referred to these publications for details of ter- minology and structural components of the basal apparatus. In this communication we used serial section postembedding immunogold electron microscopy on isolated cytoskeletons of C. reinhardtii to localize centrin in the basal apparatus. The results are illustrated in Fig. 1 to 4 and summarized in the schematic presentation of Fig. 5. In addition, we performed extensive statistical analyses of the distribution of gold particles (coated with secondary antibody) over individual basal apparatus components by comparing sections exposed to immune versus preimmune centrin IgGs (Table 1). In general, postembedding immunogold electron microscopy has the advantage over the preembedding technique that antigens inaccessible to gold particles (i.e., those located within other structural components such as basal bodies) become exposed in sections. Progress in preserving fine details of non- osmicated cellular structures and the establishment of a detailed structural map of all basal apparatus components of C. reinhardtii (13) facilitate assignment of gold particles (within the resolution determined by the size of the gold-antibody complexes) to specific structures, thus eliminating one of the traditional problems of the postembedding technique. Our preimmune controls revealed no or extremely few gold particles (Fig. 1, panels 11 and 18; Table 1), thus increasing the specificity and sensitivity of the labeling. As a critical test of this approach we investigated centrin labeling over axonemal cross sections (Table 1; Fig. 1, panels 1 to 3). About 42% of the axonemes were labeled (with an average labeling density of 1.8 gold particles/axonemal cross section; n ϭ 536). In preimmune controls, only 6% of the axonemes were labeled ( n ϭ 334). Next, we analyzed where this labeling occurred, probing 117 labeled, randomly chosen axonemal cross sections (displaying a total of 194 gold particles). We found that 96 (82%) of the cross sections showed labeling over the outer doublet microtubules and 21 (18%) were labeled in other axonemal areas (mostly over the central pair microtubules). Thus, centrin labeling in the axoneme was nonrandom and mostly confined to the outer doublet microtubules (often associated with the A tubule; Fig. 1, panels 1 to 3). No centrin labeling of the outer doublet microtubules was observed in the transitional region (the most proximal region) of the axoneme. Centrin is known as a light chain associated with one type of inner dynein arm (33, 52) (see Discussion), which is not present in this region (18). In contrast to the relatively weak anticentrin labeling in the axoneme, the star-like pattern and ring-like hub of the flagellar transitional region (well-known locations of centrin) were strongly labeled by gold particles (Fig. 1, panels 4 to 10 and 12). The statistical analysis revealed that 85% of the cross sections analyzed ( n ϭ 39) were labeled, with an average labeling density of 9.9 gold particles/transitional region (Table 1). In the preimmune control, only 3% of the transitional regions were labeled and the labeling density was low (on average 1 gold particle/transitional region). Because of the high labeling density, the distribution of label over the transitional region was not analyzed statistically. However, we noticed that the labeling was more intense over the central ring-like hub compared to the peripheral star-like pattern (Fig. 1, panels 4 to 10). In longitudinal sections of the transitional region both the distal and proximal stellate structures were labeled (Fig. 1, panels 7 to 10). Interestingly, in such sections we often encountered string-like centrin labeling extending centrally from the distal end of the distal stellate structure through the proximal stellate structure to the distal end of the basal body (Fig. 1, panels 7 to 9). The latter part of the string-like labeling corresponds to a previously identified fine filament that extends from the proximal stellate structure to a V-shaped filament system at the distal end of the basal body (13) (compare also Fig. 1, panels 8 and 9 with panels 10 and 11). The distal end of the basal body has been previously shown to contain two different filament systems, both positioned in a rotationally asymmetric manner, namely, the “acorn” and the V-shaped filament system (13) (Fig. 4, panels 37 and 38, and 5, panels 41 and 42 [schematic presentations]). The acorn, a fiber outlining the shape of an acorn in cross sections through basal bodies, is attached to triplets 7, 8, 9, 1, and 2 and is thus located in the part of the basal body facing the opposite basal body (Fig. 5, panels 39, 41, and 44). In longitudinal sections through basal bodies the V- shaped filament system is obliquely oriented (Fig. 1, panels 10, 11, and 20, and 5, panel 39). Its upper (distal) part is located at nearly the same level but opposite to the acorn and consists mainly of two 8-nm-thick filaments, which extend from microtubule triplets/doublets 4 and 5 (mostly the A tubules) into the center of the basal body lumen, where they converge, hence their V-shape in cross sections through basal bodies (Fig. ...
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Citations
... Although widely conserved in photosynthetic organisms [13,43], the molecular composition of the stellate fibers remains largely unknown. Centrin is the sole candidate identified to be part of this structure, decorating both the arms and the central cylinder of the stellate fibers [44,45]. The shared presence of Centrin in the inner scaffold raises questions about a potential common molecular composition among different microtubule connectors. ...
... These homologs (except calaxin, which is an opisthokontspecific calcium sensor for dyneins; Inaba, 2015) are also found in the genome of Ectocarpus siliculosus. Notably, centrin is involved in the reorientation of the basal body in the green alga Spermatozopsis similis (Geimer & Melkonian, 2005;Mcfadden et al., 1987) and is also localized in the flagellar basal body region of several species of brown algae (Katsaros et al., 1993). It may be a major cause of AF deflection in the male gametes of brown algae. ...
Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high‐speed recording system under different calcium conditions. The extracellular Ca ²⁺ concentration at 10 ⁻³ M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca ²⁺ concentrations below 10 ⁻² M. Real‐time Ca ²⁺ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca ²⁺ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca ²⁺ ‐dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca ²⁺ concentration.
... Our study in an early branching eukaryote reveals calciuminducible phase separation as a new centrin assembly principle ( Fig 7E). Centrins frequently associate with highly structured and polymeric cellular complexes such as the base of flagella and cilia, the inside of centrioles, the basal body and the associated contractile fibers [23,[48][49][50]]. Yet there is little evidence that centrins themselves form polymers. ...
Centrins are small calcium-binding proteins that have a variety of roles and are universally associated with eukaryotic centrosomes. Rapid proliferation of the malaria-causing parasite Plasmodium falciparum in the human blood depends on a particularly divergent and acentriolar centrosome, which incorporates several essential centrins. Their precise mode of action, however, remains unclear. In this study calcium-inducible liquid-liquid phase separation is revealed as an evolutionarily conserved principle of assembly for multiple centrins from P . falciparum and other species. Furthermore, the disordered N-terminus and calcium-binding motifs are defined as essential features for reversible biomolecular condensation, and we demonstrate that certain centrins can form co-condensates. In vivo analysis using live cell STED microscopy shows liquid-like dynamics of centrosomal centrin. Additionally, implementation of an inducible protein overexpression system reveals concentration-dependent formation of extra-centrosomal centrin assemblies with condensate-like properties. The timing of foci formation and dissolution suggests that centrin assembly is regulated. This study thereby provides a new model for centrin accumulation at eukaryotic centrosomes.
... On the one hand, fluorescence microscopy, with the use of a larger selection of better fluorophores, has made it possible to follow cellular processes and locate molecules of interest while imaging living cells. With its much higher spatial resolution, EM has allowed for studying different organisms at a much higher resolution, while obtaining information on cellular components that, combined with data from other methods, provides a valuable understanding of essential cellular processes (Cohen et al., 2002;O'Toole et al., 2002;Geimer and Melkonian, 2005;Herold et al., 2009;Kurth et al., 2010;Weiner et al., 2021;Kaplan et al., 2022). ...
The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.
... In some unicellular eukaryotes (e.g., S. cerevisiae and Chlamydomonas reinhardtii), one gene-encoding centrin was found [50,51], while in others, more than two dozen genes were identified (e.g., in Paramecium caudatum, there were found 22 genes, and in Trichomonas vaginalis, 24 genes [52]). These proteins are characterized by the presence of EF-hands motifs; each consists of two 12 amino acid EF-hand motifs that can potentially bind Ca 2+ . ...
Diatoms synthesize species-specific exoskeletons inside cells under the control of the cytoskeleton and microtubule center. Previous studies have been conducted with the visualization of the microtubule center; however, its composition has not been studied and reliably established. In the present study, several components of MTOC in diatoms, GCP (gamma complex proteins), Aurora A, and centrins have been identified. Analysis of the predicted amino acid sequences of these proteins revealed structural features typical for diatoms. We analyzed the conserved amino acids and the motives necessary for the functioning of proteins. Phylogenetic analysis of GCP showed that all major groups of diatoms are distributed over phylogenetic trees according to their systematic position. This work is a theoretical study; however, it allows drawing some conclusions about the functioning of the studied components and possible ways to regulate them.
... In addition, an important-but often neglected-feature of Chlamydomonas is the rhizoplast, which is a contractile centrin-based structure connecting basal bodies of flagella with the nuclear surface [11][12][13]. These so-called rhizoplasts or fibrous flagellar roots anchor nuclei to the flagellar or ciliar basal bodies [14][15][16][17][18][19]. The eyespot of Chlamydomonas is anchored to the D4 rootlet, extending from the peripheral flagellar basal bodies into the cell interior [20][21][22]. ...
Vision is essential for most organisms, and it is highly variable across kingdoms and domains of life. The most known and understood form is animal and human vision based on eyes. Besides the wide diversity of animal eyes, some animals such as cuttlefish and cephalopods enjoy so-called dermal or skin vision. The most simple and ancient organ of vision is the cell itself and this rudimentary vision evolved in cyanobacteria. More complex are so-called ocelloids of dinoflagellates which are composed of endocellular organelles, acting as lens- and cornea/retina-like components. Although plants have almost never been included into the recent discussions on organismal vision, their plant-specific ocelli had already been proposed by Gottlieb Haberlandt already in 1905. Here, we discuss plant ocelli and their roles in plant-specific vision, both in the shoots and roots of plants. In contrast to leaf epidermis ocelli, which are distributed throughout leaf surface, the root apex ocelli are located at the root apex transition zone and serve the light-guided root navigation. We propose that the plant ocelli evolved from the algal ocelloids, are part of complex plant sensory systems and guide cognition-based plant behavior.
... Indeed a recent study has shown that C2CD3 and LRRCC1 also localize at the lumenal distal end of the human centriole (Gaudin et al, 2022), similarly to SFI1 and Centrin, and delineate a structure reminiscent of the acorn, a filamentous density observed by electron microscopy in pro and mature Chlamydomonas basal bodies (Geimer, 2004;Gaudin et al, 2022). In addition, the acorn is accompanied by a V-shaped filament system that has been proposed to be composed of Centrin (Geimer & Melkonian, 2005). It is therefore possible that SFI1 and Centrin are part of this V-shaped filament system and associate with other distal extremity centriolar proteins, such as C2CD3 and LRRCC1, to ensure proper centriole formation and stability. ...
Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.
... In the same region, a rotationally asymmetric structure termed the 'acorn' was observed in the centriole lumen by transmission electron microscopy. The acorn appears as a filament connecting five successive triplets and is in part colocalized with Vfl1p (Geimer and Melkonian, 2005;Geimer and Melkonian, 2004). ...
Centrioles are formed by microtubule triplets in a nine-fold symmetric arrangement. In flagellated protists and in animal multiciliated cells, accessory structures tethered to specific triplets render the centrioles rotationally asymmetric, a property that is key to cytoskeletal and cellular organization in these contexts. In contrast, centrioles within the centrosome of animal cells display no conspicuous rotational asymmetry. Here, we uncover rotationally asymmetric molecular features in human centrioles. Using ultrastructure expansion microscopy, we show that LRRCC1, the ortholog of a protein originally characterized in flagellate green algae, associates preferentially to two consecutive triplets in the distal lumen of human centrioles. LRRCC1 partially co-localizes and affects the recruitment of another distal component, C2CD3, which also has an asymmetric localization pattern in the centriole lumen. Together, LRRCC1 and C2CD3 delineate a structure reminiscent of a filamentous density observed by electron microscopy in flagellates, termed the 'acorn'. Functionally, the depletion of LRRCC1 in human cells induced defects in centriole structure, ciliary assembly and ciliary signaling, supporting that LRRCC1 cooperates with C2CD3 to organizing the distal region of centrioles. Since a mutation in the LRRCC1 gene has been identified in Joubert syndrome patients, this finding is relevant in the context of human ciliopathies. Taken together, our results demonstrate that rotational asymmetry is an ancient property of centrioles that is broadly conserved in human cells. Our work also reveals that asymmetrically localized proteins are key for primary ciliogenesis and ciliary signaling in human cells.
... A member of the centrin protein family, CETN2 is a small, evolutionarily conserved, calcium-binding protein that plays an important role in humans [16,17]. Centrins are localized to centrosomes and contain two pairs of EF-hands, helix-loop-helix structures in which calcium can bind, separated by a linker region [18,19]. ...
Simple Summary
A high percentage of patients who undergo surgical resection for hepatocellular carcinoma (HCC) experience recurrence. Therefore, identification of accurate molecular markers for predicting recurrence of HCC is important. We analyzed recurrence and non-recurrence HCC tissues using two public omics datasets comprising microarray and RNA-sequencing and found novel gene signatures associated with recurrent HCC. These molecules might be used to not only predict for recurrence of HCC but also act as potential prognostic indicators for patients with HCC.
Abstract
Hepatocellular carcinoma (HCC) has a high rate of cancer recurrence (up to 70%) in patients who undergo surgical resection. We investigated prognostic gene signatures for predicting HCC recurrence using in silico gene expression analysis. Recurrence-associated gene candidates were chosen by a comparative analysis of gene expression profiles from two independent whole-transcriptome datasets in patients with HCC who underwent surgical resection. Five promising candidate genes, CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified, and the expression of these genes was evaluated using quantitative reverse transcription PCR in the validation set (n = 57). The genes CETN2, HMGA1, RACGAP1, and SNRPB, but not MPZL1, were upregulated in patients with recurrent HCC. In addition, the combination of HMGA1 and MPZL1 demonstrated the best area under the curve (0.807, 95% confidence interval [CI] = 0.681–0.899) for predicting HCC recurrence. In terms of clinicopathological correlation, CETN2, MPZL1, RACGAP1, and SNRPB were upregulated in patients with microvascular invasion, and the expression of MPZL1 and SNRPB was increased in proportion to the Edmonson tumor differentiation grade. Additionally, overexpression of CETN2, HMGA1, and RACGAP1 correlated with poor overall survival (OS) and disease-free survival (DFS) in the validation set. Finally, Cox regression analysis showed that the expression of serum alpha-fetoprotein and RACGAP1 significantly affected OS, whereas platelet count, microvascular invasion, and HMGA1 expression significantly affected DFS. In conclusion, HMGA1 and RACGAP1 may be potential prognostic biomarkers for predicting the recurrence of HCC after surgical resection.
... (A, B, D, E, I-K from Cavalier- Smith (1974 figs 5, 13, 14, 15, 17, 18, 22); C from O' Toole et al. (2003 Fig. 3F); F, H from Geimer and Melkonian (2004 fig. 1G,K), and G from Geimer and Melkonian (2005 fig. 1.8), all by permission.) ...
... I have found extensive overlooked published evidence that acorn filaments are indeed present extremely widely in eukaryotes and give further details below. Geimer and Melkonian (2004) showed that the V-filament appears as a Y at one level and V at another and later found that Vfilament components and the central filament linking them to the Chlamydomonas TP contain centrin (Geimer and Melkonian 2005) and that the centrin filaments are tilted, not strictly transverse. Whilst the centriole/TZ boundary is often assumed to lie in a single transverse plane, McNitt's (1974) serial sectioning showed the zoospore centriole of the fungus Phlyctochytrium irregulare (which Geimer and Melkonian considered to have an acorn-like structure) to be chamfered distally so doublets extend further upwards on one side than on the other. ...
... white arrows mark C tubule end, thin arrow putative thin acorn lattice; f centriolar A-B feet; N nonagonal tube in prominent centriolar distal plate (clearer in B; disrupted in A by TZ structures being pushed into centriole lumen). B inset:Chlamydomonas proximal acorn-V lattice from Geimer and Melkonian (2005) for comparison with C. C.Collodictyon TS at TF level includes centriolar nonagonal tube and grazes the putative acorn structure (doublets numbered as in Chlamydomonas (B inset). D.Collodictyon TS of cp within sleeve and surrounding stellate structure and Y links (Y). ...
I thoroughly discuss ciliary transition zone (TZ) evolution, highlighting many overlooked evolutionarily significant ultrastructural details. I establish fundamental principles of TZ ultrastructure and evolution throughout eukaryotes, inferring unrecognised ancestral TZ patterns for Fungi, opisthokonts, and Corticata (i.e., kingdoms Plantae and Chromista). Typical TZs have a dense transitional plate (TP), with a previously overlooked complex lattice as skeleton. I show most eukaryotes have centriole/TZ junction acorn-V filaments (whose ancestral function was arguably supporting central pair microtubule-nucleating sites; I discuss their role in centriole growth). Uniquely simple malawimonad TZs (without TP, simpler acorn) pinpoint the eukaryote tree's root between them and TP-bearers, highlighting novel superclades. I integrate TZ/ciliary evolution with the best multiprotein trees, naming newly recognised major eukaryote clades and revise megaclassification of basal kingdom Protozoa. Recent discovery of non-photosynthetic phagotrophic flagellates with genome-free plastids (Rhodelphis), the sister group to phylum Rhodophyta (red algae), illuminates plant and chromist early evolution. I show previously overlooked marked similarities in cell ultrastructure between Rhodelphis and Picomonas, formerly considered an early diverging chromist. In both a nonagonal tube lies between their TP and an annular septum surrounding their 9+2 ciliary axoneme. Mitochondrial dense condensations and mitochondrion-linked smooth endomembrane cytoplasmic partitioning cisternae further support grouping Picomonadea and Rhodelphea as new plant phylum Pararhoda. As Pararhoda/Rhodophyta form a robust clade on site-heterogeneous multiprotein trees, I group Pararhoda and Rhodophyta as new infrakingdom Rhodaria of Plantae within subkingdom Biliphyta, which also includes Glaucophyta with fundamentally similar TZ, uniquely in eukaryotes. I explain how biliphyte TZs generated viridiplant stellate-structures.
