A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes

Significance

Centriole/basal body (BB) connections involve two concepts: a tether and a linker. Studies have revealed essential components of the tether, while linker components remain largely unknown. Here, we describe BBLP, a trypanosome protein that is a cytoskeletal component of the linker, localizing between the BB and pro-BB throughout the cell cycle. Importantly, RNA interference (RNAi) depletion of BBLP results in a splitting of the BB–pro-BB pair subtending only the new flagellum, and perturbs mitochondrial DNA inheritance. Our data identify BBLP as the first specific protein component of the BB/centriole linker in eukaryotic cells. We provide a unifying model revealing how aspects of centriole/BB ontogeny and lineage can operate at different points of the cell cycle in evolutionarily diverse cells.

Abstract

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1–G2 tether and S–M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

Centrioles and basal bodies (CBBs) are microtubule structures found in many major eukaryotic groups; where present, they are vital for cilia and flagella formation (1) and play important roles in cell division and developmental events. CBB assembly pathways share a common set of key regulatory proteins, indicating that these structures are variations of a common pattern (2).

Faithful centriole duplication and segregation in proliferative eukaryotic cells is a well-orchestrated process (albeit with variations of pattern in different cell types across evolution) under strict temporal and spatial control and usually involve “templating”’ from a previously formed CBB (3). Two particular conceptual themes, a linker and a tether (4), have been rehearsed to explain number control, inheritance patterns, and centriole properties in mammalian cells. In interphase G1, each cell has a single centriole pair, and the duplication cycle starts in the G1/S transition and is very well described in its temporal sequence (5). During the centriole duplication and segregation cycle, centrioles are connected by the two different types of structures—the “tether” and “linker”—whose presence and disassembly at specific stages of the cell cycle are important for faithful cell-cycle progression (4, 6). The tether connects the proximal ends of the two parental centrioles from G1 to late G2 and appears important in providing a single cytoplasmic microtubule organizing center in organisms with a centrosomal architecture. Some significant studies have revealed essential components of the tether, for example CNAP and Rootletin (7, 8), Cep68 (9), LRRC45 (10), Centlein (11), and CCDC102B (12). The linker forms during S phase and connects the proximal end of the nascent procentriole to the side of the parental centriole in the orthogonal orientation. In the centriole cycle this link is described as being removed in late M phase when centriole disengagement occurs. There are two iconic features of centriole disengagement: a reorientation resulting in the loss of the original orthogonal orientation of the two paired centrioles and, second, an ability to transiently move apart (4). There is an expectation that there will be molecular components specific to each structure, but other components of the centrosome as a wider concept might play a role in both structures (4). The literature has seen a variety of terms used to describe these conceptual structures: centriole linker, centrosome linker, and so on. Here, for clarity in discussing cross-evolutionary fundamental concepts we will use the simple terms “tether” and “linker” as defined by Nigg and Stearns (4).

Current knowledge on the composition of the linker is limited, but studies in Drosophila suggest that the SAS6–ANA2 complex may play a role in centriole engagement (13). Interestingly, linker cleavage in disengagement in human cells requires the activity of the polo-like kinases and of separase, the protease responsible for sister chromatid separation (14, 15).

Many eukaryotic cells do not exhibit a centrosomally organized cytoplasm—particularly those that proliferate with assembled flagella or cilia. Although essential for cilia/flagella assembly, in such systems CBBs are often not directly involved in mitotic spindle architecture since mitosis is closed (i.e., without nuclear envelope disassembly), and anaphase and CBB separation are not concurrent. Further, in systems such as trypanosomes (16) and Leishmania (17) CBBs perform a central role in the segregation of the single mitochondrial DNA network (the kinetoplast) (18). Cell division in these organisms, where microtubule organizing centers are dispersed and do not cluster into a centrosome, involves the coordinated duplication and segregation of the nucleus (N) and the kinetoplast (K) (16). Trypanosome cells in G1 have a “1K1N” configuration, characterized by the presence of a single nucleus and a single kinetoplast, physically associated with a BB pair containing a mature BB, which subtends the flagellar axoneme, and a probasal body (pBB). At the start of S phase the pBB matures and elongates forming the new flagellum, and a new pBB forms next to each mature BB (19). The kinetoplast is divided during S/G2 (19, 20) via movement apart of the BB pairs, resulting in a characteristic “2K1N” cell, with two kinetoplasts and one nucleus. The intranuclear mitotic spindle then forms and mitosis gives rise to a cell with a “2K2N” configuration with widely separated BB pairs and associated new and old flagella. Subsequently, a mainly longitudinal cleavage furrow separates the nascent cell daughters (19).

In this study, we describe the BB linker protein (BBLP) that localizes between the BB and the pBB throughout the cell cycle. BBLP is a critical component of the linker connection between the BB and pBB, marking its initial construction at S phase and its modulation throughout that and subsequent cell cycles. This functional linker connection is compromised when RNA interference (RNAi) ablation of BBLP expression is induced and, in such knockdowns, the newly matured BB (subtending the new flagellum) becomes detached from its newly formed pBB. BBLP represents an initial insight into components that provide a cell-cycle-modulated connection between a paired mature and an immature CBB. We have analyzed our results in the light of the linker concept in mammalian cells and show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells. Further, we provide a unifying model explaining linkage differences between the two distinct centriole/BB pairs in proliferating eukaryotic cells.

Results

BBLP: A Coiled-Coil Protein that Localizes to a Cytoskeletal Structure between the BB and the pBB.

We performed a proteomic analysis of trypanosome BBs enriched by density gradient ultracentrifugation of cell lysates from a Tbkinesin II RNAi mutant with flagellarless BBs. Endogenous tagging and localization of candidates revealed a protein (Tb927.8.5400) with a unique localization and duplication pattern (Fig. 1). We refer to this protein as BBLP, based on the localization and functional data described below.

Fig. 1.

Localization of the T. brucei BBLP by fluorescence microscopy. (A) Whole cells and cytoskeletons of PCF and BSF trypanosomes expressing endogenously tagged BBLP::YFP::Ty. (B) Cytoskeletons of PCF cells expressing endogenous Ty::YFP::BBLP and Ty::Tdtomato::SAS6. In dividing cells (2K1N and 2K2N), the Ty::YFP::BBLP signal in the old flagellum BB area (arrowhead) was always stronger than that observed in the new flagellum BB area (arrow). Nuclear and mitochondrial DNA was stained by DAPI. (Scale bars, 5 μm.)

In live cells and in detergent-extracted cytoskeletons of the two main life cycle stages (the “insect form” or procyclic form [PCF] and the “mammalian form” or bloodstream form [BSF] trypanosomes) YFP::Ty-labeled BBLP (N- or C-terminus tag) had a discrete, single dot localization in the kinetoplast/BB area (Fig. 1A). SAS6, a key component of BBs/centrioles, is present throughout the trypanosome cell cycle, and in a cotagged cell line the Ty::YFP::BBLP signal appeared as a single “dot” in G1 cells (1K1N) between the two dots of Ty::Tdtomato::SAS6 (BB and pBB) (Fig. 1B). This inter-BB/pBB localization pattern remained the same throughout the cell cycle such that G2 cells (2K1N and 2K2N) had two dots of Ty::YFP::BBLP signal, each localized between a pair of Ty::Tdtomato::SAS6 dots, at the base of each of the two flagella (Fig. 1B). Importantly, in most dividing cells (90%) the signal associated with the new flagellum BB pair was weaker than that observed in the old (Fig. 1B).

BBLP has a predicted molecular weight of 108 kDa and a short N-terminal region (129 amino acid residues), followed by a large coiled-coil region (786 amino acid residues) and a short C-terminal region (26 amino acid residues). The BBLP sequence lacks Pfam-A domains, but its homologs were easily identified in other kinetoplastid protozoa, due to the conservation in the coiled-coil and C-terminal regions (SI Appendix, Fig. S1).

The inter-BB/pBB signal was seen consistently in cells expressing the N- and the C- terminal tag. In some cells expressing the C-terminal yellow fluorescent protein (YFP) tag (19% of 1K1N and 35% of 2K1N/2K2N) an additional dot of tagged BBLP was observed somewhat distal to the BB area but still associated with the proximal region of the flagellum (SI Appendix, Fig. S2). Given the high C-terminal sequence conservation it appears likely that tagging at this end is more problematic for function. We confirmed the genome identity and specificity of N-terminal tagging by immunoprecipitating Ty-tagged (BBLP::YFP::Ty) protein using the anti-Ty epitope tag BB2 antibody. By mass spectrometry, we detected peptides covering 55% of the BBLP sequence, including peptides from the first 60 amino acids.

To investigate the precise localization of BBLP we examined negatively stained and immunogold-labeled cytoskeletons expressing tagged BBLP, by transmission electron microscopy (TEM). Choosing low-density-stained material we observed gold labeling between the BB and the pBB (Fig. 2), confirming the fluorescence data. Gold labeling was often closer to the pBB, appearing not to be directly on the BBs but localized to cytoskeletal structures linking the BB/pBB pair (Fig. 2B). In dividing cells, labeling was stronger in the old flagellum BB area than in the new flagellum BB area (Fig. 2C), in agreement with the fluorescence microscopy observation (Fig. 1B).

Fig. 2.

Localization of T. brucei BBLP by immunogold electron microscopy. (A and B) TEM images of the BB area of negatively stained cytoskeletons of PCF trypanosomes expressing endogenous BBLP::YFP::Ty. Cytoskeletons were labeled with an anti–green fluorescent protein antibody and secondary antibodies conjugated to 10-nm gold. Gold particles were clearly concentrated in the area between the BB and the pBB (white arrows). (C) In dividing cells, gold particles were more numerous in the old flagellum (OF) BB area than in the BB area associated with the new flagellum (NF). (Scale bars, 200 nm.)

BBLP RNAi Leads to Organelle Positioning and Growth Defects.

To study function, we performed inducible RNAi knockdown of the BBLP protein in both PCF and BSF cells. There are subtle but interesting differences in the division process in these cell types. We observed a dramatic reduction in BBLP protein levels after RNAi induction in PCF cells (Fig. 3A). We observed a small but detectable reduction in growth after BBLP RNAi induction in PCF cultures (10% reduction from 48 h; Fig. 3B). In contrast, similar induction of BBLP RNAi led to a complete and dramatic growth arrest in BSF cells from 36 h postinduction (Fig. 3B).

Fig. 3.

Analysis of BBLP RNAi. (A) Western blotting of whole-cell lysates of PCF cells capable of inducible BBLP RNAi. Tagged BBLP (Left) was detected using BB2 antibody, while naive BBLP (Right) was detected using the antipeptide monoclonal antibody. PFR2 and TbPSA6 were used as loading controls, respectively. (B) Growth curves of PCF and BSF cells during induction of BBLP RNAi. Arrowheads mark the region of the growth curve when growth delay is first observed, after induction. (C) Counts of cells based on the number of kinetoplast masses (K) and nuclei (N) at different times postinduction of BBLP RNAi (n = 181 to 331 cells per time point). Cells with abnormal K/N ratios were those that differed from 1K1N, 2K1N, and 2K2N types.

In agreement with the growth curve data microscopy of induced cultures labeled with DAPI (4,6-diamidino-2-phenylindole) showed that the number of cells with abnormal content of DNA-containing organelles (i.e., abnormal K/N ratios, different from 1K1N, 2K1N, and 2K2N) increased after induction of BBLP RNAi (Fig. 3C), even in the PCF cell line with endogenous tagged BBLP, where no clear population growth defect was observed in that time frame. The group of cells with abnormal K/N ratios was heterogeneous, without a clear predominance of specific cell types early after induction (24 h), although 1K2N cells represented 10 to 11% of the total population at later time points postinduction (SI Appendix, Fig. S3)

BBLP RNAi Leads to “Splitting” of the New Flagellum BB/pBB Pair.

In order to understand the growth phenotype we generated a cell line with labeled BBs (endogenous Ty::YFP::SAS6) and capable of inducible BBLP RNAi. After BBLP RNAi induction these cells displayed growth defects consistent with those observed in cells with untagged BBLP or SAS6 (Fig. 3C). After 24 h of BBLP RNAi induction in PCFs, we observed that a considerable proportion of 2K1N and 2K2N cells (26% and 13%, respectively) had the posterior (new flagellum) but not the anterior (old flagellum) BB/pBB pair “split,” such that the BB and the pBB were separated by a large gap of 0.6 to 2.6 μm (Fig. 4A). In contrast, in noninduced cells (and in the BB/pBB pair associated with the old flagellum of induced cells) the BB/pBB gap was no more than 0.5 μm in length (P < 0.0001; Fig. 4 and quantified in SI Appendix, Fig. S4). This BB/pBB “splitting” phenotype was confirmed by serial-section TEM of cells where the old anterior flagellum and new posterior flagellum BB/pBB pairs could be identified based on the position of flagellum exit from the cell (Fig. 4B). In some dividing cells TEM image series show a clear separation between the new flagellum BB and pBB at the posterior of the cell (arrowheads in Fig. 4B), while the BB/pBB pair associated with the old flagellum (i.e., those from the anteriormost BB/pBB pair) remained close to each other (arrows in Fig. 4B). BSF cells displayed the BB/pBB pair splitting phenotype earlier (from 8 h) after RNAi induction (Fig. 4 A and C and SI Appendix, Fig. S4), in agreement with the earlier onset of other BBLP RNAi phenotypes (Fig. 3).

Fig. 4.

Early phenotype of BBLP RNAi. (A) As early as 8 h after induction of BBLP RNAi in both PCF and BSF trypanosomes we observed cells where the BB and the pBB (tagged with Ty::YFP::SAS6) associated with the new flagellum (white squares) were separated from each other by an unusually large gap (>500 nm). This “BB splitting” phenomenon was not observed in the new flagellum BB pair (green squares) or in induced cells, where the BB and pBB were always close to each other (<500 nm gap). DNA and flagella are labeled with DAPI and the anti-PFR2 antibody L8C4, respectively. (Scale bar, 5 μm.) (B) TEM analysis of serial sections of an induced cell, showing splitting of the new flagellum BB pair (arrowheads), as identified by the positioning and orientation of the old flagellum (arrows). (Scale bar, 800 nm.) (C) Quantification of the BB splitting phenotype at different times postinduction of BBLP RNAi (n = 500 to 636 cells per time point).

An important aspect of the BB/pBB RNAi splitting phenotype was that the BB subtending the new flagellum was always posterior relative to its split pBB, which remained closer to the unsplit anterior old flagellum associated BB/pBB pair (Figs. 4A and 5A). This configuration is likely explained by the fact that the positioning of the new flagellum BB/pBB pair—and of its associated kinetoplast—at the posterior of the dividing cell depends on the elongation and cell body attachment of the new flagellum (21, 22). We conjecture that if BBLP RNAi leads to a preferential loss (or weakening) of the linker between the posterior, new flagellum associated BB/pBB BB pair it would be expected that the pBB of this pair would be physically detached as the BB–new flagellum complex moved with growth of the posterior portion of the cell. Consequently, the “lone”’ pBB that became separated from the new flagellum upon BBLP RNAi would remain at the original (more anterior) position, closer to the anterior BB/pBB pair.

Fig. 5.

BBLP RNAi leads to a kinetoplast segregation defect. (A) Images of PCF cells before (noninduced) and after induction of BBLP RNAi for 48 h, showing BBs associated with TAC structures [identified by labeling with the anti-TAC antibody Mab22 (23)]. In induced cells, each separated BB from split BB pairs is associated with TAC components and also with a kDNA mass (Inset). (B) Quantification of the ratio between the DAPI signal intensity of daughter kinetoplasts in noninduced and induced BBLP RNAi BSF cells. *P < 0.0001 vs. noninduced 2K. At the bottom, an image of a BSF cell in the last stage of cell division, showing the daughter kinetoplasts with clearly different intensities of DAPI fluorescence. (Scale bar, 5 μm.) (C–E) TEM images of noninduced (C) and induced (D and E) cells showing a normal dividing kinetoplast (C), and abnormally large (D) and small (E) kinetoplasts observed in induced cell populations. (Scale bars, 500 nm.)

BBLP RNAi Leads to Unequal Kinetoplast DNA Segregation.

The “lone” pBB left at an anterior position after BBLP RNAi was always associated with a tripartite attachment complex (TAC) (Fig. 5A), as detected by labeling with the anti-TAC antibody Mab22 (23). The TAC physically links the BB/pBB pair to the mitochondrial kinetoplast DNA (kDNA) (18). We often observed a kDNA “dot” associated with the lone pBB in cells with the BB splitting phenotype (Fig. 5A). These dividing cells had a “3K2N” configuration of mitochondrial kDNA (never seen in normal cells), but the posterior kDNA masses appeared to have a different (and often lower) DAPI signal intensity than that of the anterior kinetoplast.

This suggested that the posterior BB/pBB splitting phenotype was generating a kDNA segregation defect, prompting us to examine kDNA segregation in dividing BSF cells after BBLP RNAi induction. We calculated the ratio between the DAPI fluorescence intensity values of the two kDNA masses in dividing cells of both induced and noninduced RNAi populations (Fig. 5B). In cells with three kDNA masses (observed in induced populations only), we calculated the DAPI fluorescence ratio between the most strongly fluorescent kDNA and the remaining two kDNA masses separately. After 24 h of RNAi induction, the ratio between the DAPI fluorescence intensities of the two daughter kinetoplasts of dividing cells was significantly higher than 1, in contrast to the expected ratio of 1 observed in noninduced dividing cells. For cells with three kDNA masses, the ratio of nearly 2 was even more asymmetric, indicating a major kDNA segregation phenotype in these cells (Fig. 5B). TEM images from induced cells also revealed asymmetric kDNA segregation, with some cells appearing to have very little kDNA mass (Fig. 5D) while others had additional, stacked kDNA networks (Fig. 5E).

BBLP Depletion Caused Flagellar Defects and Flagellar Pocket Enlargement.

Analyzing TEM images of PCF cells after 72 h of BBLP RNAi induction revealed isolated BBs that had elongated, forming transition zones, but had failed to dock on the flagellar pocket membrane surface (Fig. 6), consist with previous studies (20, 24) that docking to the flagellar pocket is not required for BB maturation in trypanosomes. Serial sections (Fig. 6D) showed that these BBs had an associated pBB. We interpret this as the “lone” pBB that, in the next cell cycle after its formation, matured and elongated into a transition zone at S phase, but because of the cellular defects was positioned incorrectly in order to locate the flagellar pocket membrane (the only area of the cell surface available for docking, as it is devoid of the subpellicular microtubule corset). Thus, docking and full elongation failed in these BBs.

Fig. 6.

BB elongation in the absence of docking, after BBLP RNAi. TEM serial section images (A–D) of a PCF cell, after induction of BBLP RNAi, showing a newly matured BB (“n”) that is 0.53 μm in length indicated that it has elongated to form a transition zone (arrow in B) (37) in the absence of docking to the flagellar pocket membrane. As a consequence, the transition zone was formed within the cytoplasm. The presence of a pBB (np in D) nearby suggests that elongation of the newly matured BB was also abortive (it should have proceeded past the transition zone). o, the old-flagellum BB; op, the pBB formed next to the old-flagellum BB. (Scale bar, 300 nm.)

In addition, BBLP RNAi resulted in an enlargement of the flagellar pocket particularly in BSF and sometimes in PCF cells (SI Appendix, Fig. S5). In BSF cultures, where 19% of cells had an enlarged flagellar pocket 24 h postinduction, of them one-sixth (∼3%) the enlarged pocket sometimes contained a coiled flagellum, emphasizing the differential architectural consequences of flagellar abnormalities in these cell types (25).

New Expressed BBLP Proteins Incorporate into Both BB Pairs Equally.

The observation that the BB/pBB splitting phenotype was only evident in the new flagellum BB/pBB pair indicated that the links between the BB and its newly formed pBB followed different inheritance patterns. One possibility was a major difference in BBLP dynamics. To examine this, we observed patterns of new BBLP incorporation into the BB/pBB pairs of cells. After short-term (2 h) inducible expression of tagged BBLP::YFP::Ty protein (Fig. 7) a BBLP signal (>40% above background) was observed in the BB/pBB area of 45% (299/659) of cells. Importantly, this included a proportion of the G1 and S cells (1K1N) (44%, 261/593) and in later G2 dividing cells (2K1N and 2K2N) (58%, 38/66), new BBLP was seen in both BB/pBB pairs—that is, those of both the new and the old flagella. In all positive 2K2N and 2K1N cells the BBLP::YFP::Ty signal intensity was similar in both BB pairs (P = 0.127; Fig. 7B). Thus, it appears that BBLP protein is dynamic and can be added to the BB/pBB linker at some point in G1/S cells and then is added to both BB/pBB pairs as the cell cycle progresses.

Fig. 7.

Induced expression of YFP-tagged BBLP in procyclic T. brucei. (A) Cytoskeletons of PCF trypanosomes were induced in tetracycline for 2 h to express BBLP::YFP::Ty. Cells in four different cell cycle stages were shown. BBs were labeled with BBA4 antibody. Nuclear and mitochondrial DNA were stained by DAPI. (Scale bar, 5 μm.) (B) The intensity of BBLP::YFP::Ty signal in old and new flagellum BB pairs of 2K1N and 2K2N cells.

Discussion

BBLP is defined by its location between a BB and a pBB. Two conceptual connections, each modulated in the cell cycle, have been rehearsed to explain centriole/BB pairs behavior: the tether and the linker (4). There are a series of key observations that indicate that BBLP is a component of the linker. All the positional information supports this conclusion; however, perhaps the most important observation is the functional aspect: when synthesis of BBLP is down-regulated by RNAi the phenotype is not that expected of a tether—the movement apart of the BB and pBB in G1 cells. Rather, the phenotype is a specific splitting of a BB and its associated pBB during later stages of the cell cycle. This is exactly what would be expected of ablation of a component of the linker.

The RNAi phenotype and localization data are, however, even more informative in that they illustrate a critical difference between the BB/pBB pairs of the old and new flagellum in the trypanosome cell cycle. BBLP is not removed from BB pairs at any point in the cell cycle (perhaps significantly, SAS6 is also maintained in trypanosome pBBs and BBs) and there is more BBLP at the BB/pBB pair of the old flagellum than at the new. Moreover, it is only the BB/pBB at the new flagellum in a proliferating cell which splits under BBLP RNAi.

Importantly, the difference in sensitivity to BBLP depletion between the old and new flagellum BB/pBB pairs suggests that the pattern of centriole/BB linker formation and inheritance in trypanosomes exhibits a level of complexity not reported in mammalian cell models. In mammalian cells, the linkers are envisaged to be assembled de novo upon new centriole formation, because the old linkers are disassembled during centriole disengagement in late mitosis of the previous cell cycle (4). In trypanosomes, the stronger BBLP signal in the old flagellum BB pair, together with the apparent lack of BB splitting in this BB pair upon BBLP RNAi, suggests that the linker in the old flagellum is different from that found in the new flagellum, despite the fact that these links are likely to be established concomitantly, when new pBBs are formed (Fig. 8).

Fig. 8.

Models of centriole and BB linker in mammalian and trypanosome cell cycles. (Top) Mammalian cycle based on Nigg and Stearns (4). The tether is represented in blue in the mammalian cycle. We, as yet, have no information on the tether structure in the trypanosome cell cycle and hence do not illustrate it in the cartoon. The linker (red) is formed in S phase, when two procentrioles (3, 4) are formed by the side of their parent centrioles (1, 2). The linkers persist until the end of mitosis, when a process called disengagement occurs, leading to the loss of orthogonal orientation of procentrioles to their parent centriole, and their ability to separate. (Bottom) Trypanosome cycle based on Woodward and Gull (38) The linkers are formed in S phase. We envisage two construction modes for linker 1:3 and 2:4 since this is the first time that BB2 has formed a pBB and a linker while BB1 has formed these structures previously in its history. Hence, we introduce the concept of centriole/BB specific linkers given the lineage history and maturation state of the centriole/BB. In the trypanosome cell cycle the two aspects of disengagement, loss of orthogonal orientation and ability to move apart, occur at separate times. Loss of orthogonal orientation of pBBs occurs in mid-G2 onward; however, the BB/pBB pairs (1:3 and 2:4) are still intimately connected. They acquire freedom to move apart in late G1/early S phase of the next cell cycle when BB2 matures, elongates, and docks with the flagella pocket membrane.

Our induction experiment shows that new BBLP is added to the linkers of both the old and new flagellum BB/pBB pairs, illustrating that both are dynamic within the proliferative cell cycle. Evidence from cell-cycle analyses of genome-wide transcript and protein levels shows that the BBLP transcript is a late-G1-expressed transcript but the overall protein level is unchanging (26). This would fit with a model whereby the BBLP expression is switched on at the start of the cell cycle in readiness for the formation of the new linkers associated with the old and new flagella.

The higher amount of BBLP at the BB/pBB pair of the old flagellum and the insensitivity of this linker to RNAi ablation suggests an intrinsic lineage difference between the two BB pairs in the cell. It is well known that there are maturation differences in centriolar appendages between centriole pairs in G2 mammalian cells, and evidence in trypanosomes also shows that BBs have a defined maturation lineage across multiple cell cycles (27) distinguishing the two BB pairs in a dividing cell. This informs a more sophisticated view of the linker between centrioles or BB pairs. We sought to unify this view in both the BB and centriole biology of proliferating cells. In mammalian cells the linker function is removed at the end of mitosis in a process known as centriole disengagement which exhibits two aspects: loss of centriole orthogonal orientation and their ability to separate and move apart. The loss of orthogonal orientation is a defined process that happens in mid-G2 onward in the trypanosome cell cycle—characteristic of cells where the centrioles/BBs are not associated with the establishment of bipolarity by a mitotic spindle. We can place the other aspect of disengagement at an earlier different point in the cell cycle—late G1 to early S. It is at this point that the pBB matures, extends a transition zone and flagellum, and begins to move away from its parent BB to divide the flagellar pocket (28). In Fig. 8 we compare these events in the two evolutionarily diverse cell types and introduce the concept that there is a distinct difference between the linker constructed between BB1/pBB3 and BB2/pBB4 in the cell cycle. The model in trypanosomes suggests that an original, mature linker in G1 cells is asymmetrically “severed” at the centriole separation aspect of disengagement. Thus, more BBLP stays with BB1 than BB2 and this asymmetry is maintained as the new pBBs form. The linker forming on BB1 is more mature than the linker forming on BB2, which is the first time that this BB has formed a linker to template a new pBB. Consolidation of the linkers continues through G2 but differences are maintained (amount of BBLP and sensitivity to RNAi insult) until late in the cycle when they reach maturation at cytokinesis.

The RNAi phenotype, together with the localization data, supports the conclusion that BBLP is a key component of the linker between BB and pBB established upon new pBB formation, early in the S phase of cell division cycle. However, our results indicate that during disengagement linker components, of which BBLP is one, are not degraded entirely but are asymmetrically inherited, perhaps indicating a mix of proteolytic and posttranslational modifications of different components.

Analyzed of our results in light of the linker concept in mammalian cells shows that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent, but separable, events in evolutionarily diverse cells. Further, our unifying model emphasizes linkage differences between the two different centriole/BB pairs in a proliferating cell. We envisage that this pattern of diversity in linker lineage is likely to be seen in centrioles of other cells when more is known of their components.

Materials and Methods

Trypanosome Cell Culture and Transfections.

Trypanosoma brucei brucei SmOXP927 (29) were cultured at 28 °C in SDM 79 medium supplemented with 10% fetal calf serum (FCS). T. brucei SmOXB427 (29) were cultured at 37 °C with 5% CO₂ in HMI-9 medium supplemented with 10% FCS. All cell lines were transfected as described previously (29). Cell growth was monitored using a CASY1 cell counter (Schärfe System GmbH).

Endogenous Tagging, Inducible Expression, and RNAi.

To produce the pEnt6B-BBLP-3′YFP-TY cell line, expressing endogenously tagged BBLP::YFP::Ty, the 3′ end of the BBLP coding sequence (Tb927.8.5400, 390 base pairs [bp]) and 3′ untranslated region (UTR) (475 bp) were amplified using the primers BBLP-3S-NotI, BBLP-3A-SpeI, BBLP-3US-HindIII, and BBLP-3UA-NotI (SI Appendix, Table S1) and cloned into SpeI and HindIII sites of pEnt6B-Y (30). To produce the pEnt6B-BBLP-5′YFP-TY cell line, expressing endogenously tagged Ty::YFP::BBLP, the 5′ UTR (321 bp) and 5′ end of the BBLP coding sequence (620 bp) were amplified using the primers BBLP-5US-XhoI, BBLP-5UA-BamHI, BBLP-5S-XbaI, and BBLP-5A-XhoI (SI Appendix, Table S1) and cloned into BamHI and XbaI sites of pEnt6B-Y. To generate the “double tag” cell line expressing Ty::YFP::BBLP and Ty::TdTomato::SAS6 (pEnt6BN-Tdtomato-SAS6-YFP-BBLP), the pEnt6N-Tdtomato vector was generated from pEnt6B-Y by replacing the blasticidin resistance and YFP genes with neomycin resistance and Tdtomato genes, respectively. Initially, the blasticidin resistance gene was removed using EcoRI and NcoI sites, and the neomycin gene (amplified from p2705IFT using the primers of EcoRIS-NeoR and BspHIA-NeoR; SI Appendix, Table S1) was ligated into the pEnt6 backbone, to generate pEnt6N-Y. Then, the Tdtomato gene was removed from pBA148 (31) and inserted into pEnt6N-Y using SpeI XbaI sites, to generate pEnt6N-Tdtomato. SAS6 5′ UTR and coding sequences were cut from pEnt6PY-SAS6 and ligated into pEnt6N-Tdtomato, generating pEnt6N-SAS6-5′-Tdtomato-TY (for 5′ tagging of SAS6). Then, NotI-linearized pEnt6N-SAS6-5′Tdtomato-TY was transfected into the pEnt6B-BBLP-5′YFP-TY cell line, for endogenous tagging of SAS6 with TdTomato.

To generate the cell line with inducible BBLP-YFP expression (pDex577-BBLP-YFP), the full-length BBLP coding sequence was amplified from genomic DNA using Dlai927.8.5400S-HindIII and Dlai927.8.5400A-SpeI primers and cloned into the HindIII and SpeI sites of pDex577 (30). Tetracycline (1 μg/mL) was added to culture medium for 2 h to induce BBLP::YFP::Ty expression.

To generate cell lines capable of inducible BBLP knockdown by RNAi, a 461-bp fragment of the T. brucei BBLP coding sequence (Tb927.8.5400) (23) was amplified by from genomic DNA (see PCR primer sequences in SI Appendix, Table S1) and cloned into the BamHI and HindIII sites of the inducible RNAi vector p2T7-177 (32). Then, the following cell lines were transfected with the BBLP-p2T7-177 construct: the pEnt6B-BBLP-3′YFP-TY cell line (PCF), as well as SmOXP927 and SmOXB427. To generate inducible BBLP knockdown in cells cells with YFP-tagged SAS6, the linearized pEnt6PY-SAS6 plasmid was transfected into BBLP-p2T7-177 cells on SmOXP927 and SmOXB427 backgrounds. The tagged protein also contained a short Ty-1 tag, which could be detected using the monoclonal antibody BB2 (33, 34).

To induce BBLP RNAi, BBLP-RNAi cell lines were grown in culture media supplemented with 1 μg/mL doxycycline for up to 72 h.

Fluorescence Microscopy Analyses.

For immunofluorescence and K/N counts, PCF cells were washed in phosphate-buffered saline (PBS) and settled onto superfrost slides (Menzel-Gläser; Thermo), while BSF cells were pelleted, resuspended in FCS, and smeared and dried onto microscope slides. Cells on slides were fixed/permeabilized by immersion in cold methanol (−20 °C) for 5 min. After rehydration in PBS, cells were mounted with coverslips using DABCO antifade medium containinig DAPI.

Alternatively, rehydrated slides containing PCF cells capable of inducible BBLP RNAi and expressing Ty::YFP::SAS6 were blocked in 5% bovine serum albumin in PBS for 30 min and labeled with the Mab22 monoclonal antibody [neat supernatant (35)] for 1 h. Then, slides were washed in PBS and incubated in anti–immunoglobulin M-TRITC secondary antibodies (AP128R from Millipore; diluted 1:200) for 1 h. After final PBS washes, slides were stained in DAPI and mounted in DABCO as described above.

All slides were observed in a Zeiss Axioplan 2 or Leica DM6500 microcope, and images were analyzed using the ImageJ software. A total of 500 cells per time point were scored manually for their K and N content. For kDNA intensity analysis, each fluorescent kDNA spot observed in the DAPI channel was enclosed in a 10-pixel-radius circle, whose center was located at the point of highest signal intensity. For individual kinetoplasts, kDNA fluorescence intensity values represented the total fluorescence intensity for the circular area, subtracted of a background value defined as the lowest intensity value within the circle in cells with more than one kinetoplast. Data were organized and presented in GraphPad Prism 5, and statistical analysis was performed using the χ² test.

Bioinformatics.

Orthologs of the T. brucei BBLP in other species were identified using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/) and hidden Markov models (http://hmmer.janelia.org/). Kinetoplastid homologs of the T. brucei proteins were identified by reciprocal BLAST against three kinetoplastid genomes (Trypanosoma cruzi, Leishmania major, and Leishmania braziliensis from https://tritrypdb.org/tritrypdb/app), using a 1E-6 e-value cut off. The existence of coiled-coil regions in BBLP proteins was evaluated using the COILS server (36).

Other methods used in this paper can be found in SI Appendix, Materials & Methods.

Data Availability

All study data are included in the article and/or SI Appendix.