TtBld10 is a conserved basal body cartwheel outer domain protein
The outer cartwheel domain protein Poc1 stabilizes basal bodies (Pearson et al., 2009b
). Because CBB stability is essential for its function, we searched for other outer cartwheel proteins that act as basal body stability factors. Bld10/Cep135 was a good candidate because it also localizes to the outer cartwheel domain of Chlamydomonas
basal bodies (Hiraki et al., 2007
; Jerka-Dziadosz et al., 2010
). A single BLD10/CEP135
orthologue exists in the Tetrahymena thermophila
genome and will be referred to as TtBLD10
encodes a 171-kDa protein, TtBld10. As with other Bld10 family members, the protein contains extensive coiled-coil domains with two conserved regions called conserved region 1 (CR1) and conserved region 2 (CR2; Carvalho-Santos et al., 2010
; Hodges et al., 2010
; Supplemental Figure S1A). TtBld10 shares 42% protein sequence similarity with the human Bld10 homologue, Cep135 (Supplemental Figure S1, B and C). Consistent with a role in basal body assembly, TtBLD10
is expressed similarly to other core Tetrahymena
basal body components (Miao et al., 2009
We localized TtBld10 to determine whether TtBld10 shares a similar localization profile to that observed in other organisms. TtBld10-mCherry was expressed under the control of its native TtBLD10
promoter in Tetrahymena
cells. We found that TtBld10-mCherry localizes with TtCen1 (Stemm-Wolf et al., 2005
) at all basal bodies and remains localized to basal bodies at all stages of the cell cycle (). Moreover, TtBld10-mCherry did not localize in cilia (). Similar to other organisms tested, TtBld10 is a CBB protein.
FIGURE 1: TtBld10 localizes to the basal body outer cartwheel domain. (A) Both TtBld10 and TtCen1 colocalize at basal bodies. T. thermophila cells expressing TtBld10-mCherry (red) were stained for the basal body marker TtCen1 (green; Stemm-Wolf et al., (more ...)
To determine where TtBld10 localizes within the basal body architecture, we colocalized TtBld10-mCherry relative to TtCen1, which localizes asymmetrically to the proximal end and to the site of kinetodesmal fiber attachment of basal bodies (Stemm-Wolf et al., 2005
). TtBld10 localized to the proximal end of the basal body, coincident with the site of the cartwheel (), and was not found along the length of the basal body. We then localized TtBld10-mCherry relative to green fluorescent protein (GFP)–TtSas6a, which localizes to the central hub of the cartwheel (Kilburn et al., 2007
; Supplemental Figure S1D). TtBld10 localizes peripherally to TtSas6a, consistent with its localization to the outer cartwheel domain. Next, we used immuno–electron microscopy (IEM) to determine the ultrastructural localization of TtBld10-GFP. Consistent with our fluorescence data, the majority (73%) of TtBld10 immuno-gold label localized to the basal body cartwheel (). We found that a small fraction (14%) of TtBld10 localizes to the terminal plate (). Drosophila
Bld10 localizes to the distal end of basal bodies and is required to form the central doublet microtubules of motile cilia (Blachon et al., 2009
; Mottier-Pavie and Megraw, 2009
; Carvalho-Santos et al., 2010
). This raises the possibility that TtBld10, like DmBld10, may be required for central doublet formation. However, axoneme central doublet microtubules were normal in Ttbld10Δ
cells (unpublished data), suggesting that TtBld10 does not regulate the axoneme central pair microtubules in Tetrahymena
as it does in Drosophila.
To determine where TtBld10-GFP localizes within the cartwheel, we quantified the relative immuno-gold distribution in cross-sectional views of the cartwheel. TtBld10 associates with the ends of the cartwheel spokes (39%) and triplet microtubules (44%; ). This is consistent with Chlamydomonas
Bld10, which also localizes to the outer cartwheel and spoke tips (Hiraki et al., 2007
; Jerka-Dziadosz et al., 2010
). Taken together, the results indicate that TtBld10 localization is predominantly restricted to the basal body outer cartwheel.
Ttbld10Δ causes the loss of basal bodies
Prior studies addressing the function of Bld10/Cep135 were limited to using hypomorphic alleles and knockdowns because a complete BLD10
genomic knockout was not accessible. Here we created, for the first time, a complete genomic knockout of BLD10
was induced by mating two Ttbld10Δ
heterokaryon knockout strains to produce progeny with complete macronuclear Ttbld10Δ
(Hai et al., 2000
). Control cells were generated by mating wild-type cells with either heterokaryon knockout strain, which results in phenotypically normal cells. These control cells are referred to as TtBLD10
cells exhibit deleterious phenotypes that are common among basal body and ciliary mutants (Brown et al., 1999
; Pearson and Winey, 2009
causes cellular lethality. To determine the number of cellular divisions that Ttbld10Δ
cells underwent before death, we quantified growth rates of Ttbld10Δ
cell populations. Ttbld10Δ
cells averaged 3.1 ± 0.7 divisions before division ceased (n
= 3; Supplemental Figure S2). In addition to a reduced rate of cellular growth, the qualitative rate of cellular swimming was reduced in Ttbld10Δ
cells. Moreover, Ttbld10Δ
cells exhibited a decrease in directed forward motility, as seen by an increase in lateral cellular movement relative to forward movement. In summary, TtBld10 is required for cell viability, motility, and normal cell cycle progression.
cells exhibited similar, albeit stronger, mutant phenotypes compared with Ttpoc1Δ
cells, we next asked whether TtBld10 loss, like TtPoc1 loss, affects the total number of basal bodies per cell (Pearson et al., 2009b
). We quantified the frequency of α-TtCen1–stained basal bodies at 0, 12, 24, and 48 h after TtBLD10
knockout. Basal body number per cell declined and organization was increasingly disrupted with time after TtBLD10
knockout (, A and C). Moreover, the basal bodies of the oral apparatus disassembled in Ttbld10Δ
cells (). To confirm that the loss of TtBLD10
was responsible for the observed phenotypes, we rescued the Ttbld10Δ
cells by reintroducing the wild-type TtBLD10
gene after knockout. Basal body number and organization were both restored by the reintroduction of TtBLD10
(). Thus TtBld10, like other basal body components, is required for normal basal body frequency and organization (Stemm-Wolf et al., 2005
; Culver et al., 2009
; Pearson et al., 2009b
; Pearson and Winey, 2009
FIGURE 2: TtBLD10 knockout causes a loss of basal bodies. (A) TtBld10 is required to maintain the normal number and organization of basal bodies. Images describe a time course of basal bodies after Ttbld10Δ. Basal bodies are visualized by anti-TtCen1 staining. (more ...)
Bld10 is required for new basal body assembly
The inhibition of new basal body assembly causes a progressive reduction of basal bodies at each cell division. This is because basal bodies are segregated to the future cells without producing new ones to maintain the normal complement of basal bodies. In Tetrahymena
, new basal bodies form anteriorly to existing basal bodies. These basal bodies form as doublets after assembly (one new and one old). The newly assembled basal body then moves anteriorly away from the old basal body while maturing into a basal body that nucleates a cilium (Allen, 1969
; Ng and Frankel, 1977
). To determine whether new basal bodies are formed in Ttbld10Δ
cells, we visualized both old and new basal bodies. Old basal bodies were labeled with a marker that surrounds mature basal bodies that resembles the K-antigen (Williams et al., 1990
), here called K-like antigen (Kl-Ag). This is colocalized with the panspecific basal body marker centrin (TtCen1; Stemm-Wolf et al., 2005
). Kl-Ag levels increase with basal body maturity (Supplemental Figure S3A). New basal body assembly is evident as basal body doublets with TtCen1 staining but no Kl-Ag staining at the anteriorly positioned basal body (, A and B). Approximately 18% of the total basal bodies were newly duplicated at both 0 and 24 h for control cells (; green arrow). The proportion of newly assembled basal bodies dramatically decreased from 18% (0 h) to 3% (24 h) in Ttbld10Δ
cells (). Moreover, we were unable to identify new basal body assembly in Ttbld10Δ
cells at later time points (36 and 48 h). We predict that the basal body assembly observed in Ttbld10Δ
cells (0, 12, and 24 h) is the result of residual, yet reduced, TtBld10 protein after knockout. The amount of new assembly decreases and is not detectable by 36 h. Thus TtBld10 is required for new basal body assembly.
FIGURE 3: TtBld10 is required for basal body assembly. (A) Fluorescence images of all basal bodies (anti-TtCen1; green) and mature basal bodies (Kl-Ag; red) in control and Ttbld10Δ cells 12 h after knockout. Newly assembled basal bodies are evident as the (more ...)
Of interest, we observed Kl-Ag–stained foci without TtCen1 staining in Ttbld10Δ
cells (, red arrowhead). The original K-Ag antibody recognizes domains within the membrane skeleton surrounding basal bodies but does not directly stain basal bodies (Williams et al., 1990
). Kl-Ag accumulates at these sites with time after basal body assembly and remains at these sites even in the absence of basal bodies in cycling cells (, red arrowhead, and Supplemental Figure S3A). Thus loss of basal bodies does not result in the loss of Kl-Ag staining in cycling cells. We find Kl-Ag staining in the absence of TtCen1, and these foci mark locations where basal bodies once existed and are now sites of basal body disassembly. Furthermore, basal body disassembly occurred at immature basal bodies, as judged by the reduced level of Kl-Ag staining relative to Kl-Ag levels in mature basal bodies. This suggests that the basal bodies disassembled before their complete maturation. TtBld10 is, therefore, required not only for new basal body assembly, but also to stabilize developing basal bodies.
Bld10 is required to stabilize and maintain basal bodies
Basal bodies in G1-arrested cells have full levels of Kl-Ag, which indicates that they are mature. We tested whether mature basal bodies (as judged by Kl-Ag) disassemble in the absence of TtBld10. Ttbld10Δ cells were arrested in G1, so that cell division and new basal body assembly was repressed. A reduced number of basal bodies was observed in G1-arrested Ttbld10Δ cells compared with control cells (, A and B). Moreover, the decrease in basal body number was time dependent, suggesting that basal bodies did not immediately disassemble, but instead there was a temporal loss in basal bodies. These results further indicate that TtBld10 has an important role in maintaining and stabilizing existing basal bodies.
FIGURE 4: Bld10 is necessary for the maintenance of basal bodies. (A) Basal bodies disassemble in G1 cell cycle–arrested Ttbld10Δ cells. Immunofluorescence images of cell cycle–arrested Ttbld10Δ cells stained for the basal body marker (more ...)
To directly visualize basal body disassembly, we used Kl-Ag to mark the site of basal bodies that existed before TtBld10 knockout. We colocalized Kl-Ag with TtCen1 in Ttbld10Δ cells that were arrested in G1. Disassembly events (Kl-Ag staining without TtCen1 staining) in Ttbld10Δ cells were observed in a low but significant (p < 0.001) fraction of the basal body pool relative to TtBLD10 cells (, C and D). We hypothesize that this low fraction is due to a transient Kl-Ag signal after basal body disassembly in G1-arrested cells, and this makes disassembly events difficult to capture. Moreover, the progression of Kl-Ag disassembly was visualized in G1-arrested Ttbld10Δ cells (Supplemental Figure S3B). Basal body and Kl-Ag disassembly was not observed in control cells. Thus TtBld10 is required to maintain both immature and mature basal bodies.
TtBld10 promotes triplet microtubule stability
Because TtBld10 is required for the assembly of new basal bodies and the stability of existing basal bodies, we hypothesized that TtBld10 regulates the core CBB structure. In particular, we postulated that TtBld10 regulates the triplet microtubules that comprise CBBs. The A-tubules of triplet microtubule blades are attached to the central hub of the cartwheel via a spoke linkage. After assembly and attachment of the A-tubule to the cartwheels, the B- and C-tubules are then sequentially added (Dippell, 1968
; Guichard et al., 2010
). To determine whether this organization is affected by TtBld10 loss, we visualized the basal body ultrastructure in Ttbld10Δ
cells at 12 h postknockout were prepared for transmission electron microscopy (TEM; Dahl and Staehelin, 1989
; Meehl et al., 2009
; Winey et al., 2012
). Seventy-one percent of the Ttbld10Δ
basal bodies exhibited defects that were not found in control basal bodies (; n
= 100 basal bodies). The Ttbld10Δ
-associated defects in triplet microtubules were categorized into three classes. Seventy-four percent of the microtubule-defective Ttbld10Δ
basal bodies were missing a single or multiple tubules of the microtubule triplet blade, causing basal bodies to have only doublet or singlet microtubules in at least one of the basal body triplet microtubule positions (, class 1). In cases in which a doublet was present instead of a triplet the C-tubule was missing most commonly (65% of class 1 mutants); however, a significant fraction of A-tubules were also missing (22% of class 1 mutants). In cases in which a singlet was present instead of a triplet the B-and C-tubules were always missing. In 56% of class 1 basal bodies, the missing tubule was lost from the entire basal body length based on serial sections. In the remaining class 1 samples, the basal body proximal end contained all three tubules of the microtubule triplet, and the segment distal to the cartwheel exhibited a decreased number of tubules, generating doublet or singlet morphology. The instability of tubules of the basal body triplet (class 1) was the major defect found in Ttbld10Δ
FIGURE 5: Triplet microtubule assembly and stability defects in Ttbld10Δ cells. (A) Electron micrograph image of a cross-sectional view of a TtBLD10 control cell (top). Middle, representative schematic of the basal body structure. Bottom, relative frequency (more ...)
Fifteen percent of defective Ttbld10Δ
basal bodies were missing at least one triplet microtubule through the entire length of the basal body (, class 2). A similar basal body phenotype was found in Paramecium
cells where PtBld10a was depleted by RNA interference (Jerka-Dziadosz et al., 2010
). The Tetrahymena
phenotype was separated into two subcategories. Class 2a consists of basal bodies with triplet microtubules that conform to the missing gap, thereby decreasing the basal body diameter (, class 2a; 10% of defective basal bodies). Class 2b consists of a missing triplet microtubule that produces a gap in the ninefold symmetry (, class 2b; 5% of defective basal bodies). In class 2a, the ninefold symmetry was likely never established, and this represents a basal body assembly defect. In class 2b, the missing triplet microtubule likely established correct ninefold symmetry; however, microtubule attachment and stability was disrupted.
The third class of Ttbld10Δ basal body defects (class 3; 11% of defective Ttbld10Δ basal bodies) consisted of a combination of the first two classes, in which tubules of microtubule triplets and complete triplets are missing (). Thus triplet microtubule stabilization and organization are lost in Ttbld10Δ cells.
In addition to disruption of the individual triplet microtubule structure, we find that the majority (56%) of basal bodies in Ttbld10Δ cells display triplet microtubule orientation defects. These defects are characterized by off-axis positioning of entire triplet microtubule blades (). Moreover, 77% of basal bodies that possess class 1–3 phenotypes also exhibit triplet microtubule orientation defects. These defects, in conjunction with the triplet microtubule structural defects (classes 1–3), suggest that TtBld10 stabilizes the structure and orientation of the basal body triplet microtubules.
TtBld10 protein stably incorporates during basal body assembly and maturation
The disassembly of both immature (new, daughter) basal bodies in Ttbld10Δ cycling cells and mature (old, mother) basal bodies in Ttbld10Δ G1-arrested cells led us to ask when TtBld10 is incorporated at basal bodies to perform its functions. We quantified the relative amounts and the timing of when TtBld10 protein incorporates during the assembly of new basal bodies and the maintenance of existing basal bodies. TtBld10-mCherry levels were variable, depending on the age of the basal body. The basal body age was estimated based on distance between the daughter and mother basal bodies. Newly assembled, daughter basal bodies were closely positioned near the mother basal body, whereas older daughter basal bodies were more physically separated from their mother basal bodies. Mature basal bodies had increased levels of TtBld10 protein compared with immature basal bodies (, A and B). At the time when the separation of mother and daughter basal bodies can be resolved, newly assembled basal bodies have a mean Bld10-mCherry fluorescence intensity of ~40% of the mother TtBld10-mCherry fluorescence intensity. As the basal body increases in separation from its mother, an increased level of TtBld10-mCherry is observed until a maximum protein level is reached that is equal to that of the mother basal body. Mature basal bodies did not increase or decrease in fluorescence with time, suggesting that once the basal body reaches the maximum level of TtBld10-mCherry, this level remains constant (). A similar incorporation behavior was observed using GFP-TtBld10 (Supplemental Figure S4, A and B). Thus TtBld10 protein levels accumulate as basal bodies temporally mature.
FIGURE 6: TtBld10 protein stably accumulates at basal bodies. (A) TtBld10-mCherry fluorescence intensities are low at newly formed basal bodies and increase as the daughter basal bodies separate from their mother basal bodies. The separation distance corresponds (more ...)
Fluorescence recovery after photobleaching (FRAP) was used to determine whether the incorporated TtBld10 protein is stably associated with basal bodies. Single, TtBld10-mCherry–labeled mature basal bodies were photobleached, and live-cell imaging was used to visualize the kinetics of protein redistribution and fluorescence recovery. A low level of TtBld10-mCherry fluorescence recovery was observed, indicating that TtBld10 is stably bound to the basal body (percentage recovery, <5%; ). When we performed longer recovery experiments of up to 10 min, we also did not observe a significant fluorescence recovery (unpublished data). This supports the model that once TtBld10 protein incorporates, it remains at basal bodies.
To determine whether TtBld10 levels were recruited to maximum levels before ciliogenesis, we quantified the levels of TtBld10-mCherry at basal bodies possessing a cilium. TtBld10 protein is at its maximum level in ciliated basal bodies (, arrows). This suggests that only mature basal bodies, as judged by TtBld10 levels, produce a cilium. Because mature basal bodies disassemble in arrested Ttbld10Δ cells, we hypothesized that disassembly is due to ciliary beating. The instability of basal bodies without TtBld10 led us to two simplified models for TtBld10 function. First, TtBld10 is required early during new basal body assembly for assembly and stabilization. Second, TtBld10 stabilizes mature basal bodies to resist forces generated by beating cilia.
Decreased ciliary beating rescues basal body instability in Ttbld10Δ cells
TtBld10 loss causes mature basal bodies to disassemble, and ciliated basal bodies have a maximum level of TtBld10 protein (, A and B, and ). This suggests that TtBld10 is required to resist the forces created by ciliogenesis or ciliary beating. We assessed whether ciliary beating promotes the disassembly of basal bodies in Ttbld10Δ cells. Tetrahymena cells use cilia-dependent forces to move. To test whether Ttbld10Δ basal bodies disassemble as a result of the forces produced by ciliary beating, we inhibited ciliary beating in G1 cell cycle–arrested Ttbld10Δ cells at 12 and 24 h post–TtBLD10 knockout by treatment with NiCl2. We found that inhibition of ciliary beating significantly rescued basal body frequency and organization in Ttbld10Δ cells compared with control cells (). These data suggest that TtBld10 stabilizes basal bodies to resist cilia-dependent forces.
FIGURE 7: Cilia-generated forces destabilize basal bodies in Ttbld10Δ cells. (A) Ciliary inhibition rescues basal body disassembly in Ttbld10Δ. G1-arrested TtBLD10 and Ttbld10Δ cells at 12 and 24 h postknockout and treated with NiCl2 to (more ...)
In addition to inhibiting ciliary beating, we increased the physical resistance or drag force of the media in which cells swim by increasing the media viscosity with 5% polyethylene oxide (PEO). G1 cell cycle–arrested Ttbld10Δ cells in 5% PEO disassemble basal bodies at a significantly greater level compared with Ttbld10Δ cells swimming in normal media (). The frequency of basal bodies was not affected in control cells treated with 5% PEO, suggesting that TtBld10 is necessary to stabilize basal bodies from the increased drag force.