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Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

['Charlotte Isch', 'Univ. Bordeaux', 'Cnrs', 'Microbiologie Fondamentale Et Pathogénicité', 'Umr', 'Bordeaux', 'Paul Majneri', 'Max Perutz Labs', 'Vienna Biocenter', 'Medical University Of Vienna']

Date: 2021-08

The flagellar pocket (FP) is the only endo- and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 Å resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.

Here we identify another bona fide FPC protein, BILBO2, so named because of close similarity with BILBO1 in protein localization and functional domains. We demonstrate that BILBO1 and BILBO2 share a common N-terminal domain involved in the interaction with FPC4, and illustrate a tripartite interaction between BILBO1, BILBO2, and FPC4. Our study also provides the first atomic view of two FPC components. These data represent an additional step in deciphering the FPC structure and function in T. brucei.

Trypanosomes belong to a group of zoonotic, protist, parasites that are found in Africa, Asia, South America, and Europe and are responsible for severe human and animal diseases. They all have a common structure called the flagellar pocket (FP). In most trypanosomes, all macromolecular exchanges between the trypanosome and the environment occur via the FP. The FP is thus essential for cell viability and evading the host immune response. We have been studying the flagellar pocket collar (FPC), an enigmatic macromolecular structure at the neck of the FP, and demonstrated its essentiality for the biogenesis of the FP. We demonstrated that BILBO1 is an essential protein of the FPC that interacts with other proteins including a microtubule-binding protein FPC4.

Funding: This work was supported by the CNRS and the University of Bordeaux to DRR and MB, the LabEx ParaFrap [ANR-11-LABX-0024] to DRR, the Max Perutz Labs and grant [P24383-B21] and [I4960-B] from the Austrian Science Fund (FWF) to GD, the ANR-FWF PRCI [ANR-20-CE91-0003] to MB. YP was supported by the “Integrative Structural Biology” PhD program [W-1258 Doktoratskollegs] funded by the FWF and CI was supported by the LabEx Parafrap PhD program [ANR-11-LABX-0024]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The numerical data used in Figs 3D and 4D are included in S1 Data . Coordinates and structure factors of the crystal structure of the BILBO2-NTD/FPC4-CTD complex have been deposited in the Protein Data Bank (PDB) under accession code 7a1i. https://doi.org/10.2210/pdb7A1I/pdb .

Copyright: © 2021 Isch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Despite extensive structural and functional studies on BILBO1, the mechanisms underlying the macro-molecular assembly and biogenesis of the FPC remain elusive, mainly due to the poor knowledge of its molecular composition and assembly. Here we report the identification and characterization of Tb927.6.3240, a novel BILBO1-partner protein at the FPC. We show that it is a bona fide FPC protein with an N-terminal domain structurally and functionally homologous to that of BILBO1. We thus named it BILBO2. We demonstrate here that similarly to BILBO1, BILBO2 interacts with the CTD of FPC4 via its NTD. We have further determined a 1.9-Å resolution crystal structure of the BILBO2-NTD/FPC4-CTD complex, which provides a clear view of how the extended FPC4 polypeptide docks into the horseshoe-like aromatic pocket of BILBO2. This is the first molecular structure of the FPC protein complex and represents a significant step toward deciphering the FPC interactome in T. brucei. Overall, our data identify a common module in two different FPC components that are essential in FPC biogenesis and cell viability.

To date, a handful of FPC or FPC-associated proteins have been identified (BILBO1, FPC5, FPC4, Tb927.11.5640) [ 4 , 9 , 15 , 16 ] but only two, BILBO1 and FPC4, have been characterized at the molecular level [ 4 , 10 , 13 , 15 ]. We have previously demonstrated that FPC4 binds to microtubules via its N-terminus, and interacts with the NTD of BILBO1 via its C-terminal domain (CTD). In T. brucei, FPC4 localizes at the interface between the FPC and the HC, suggesting a role in linking the FPC-HC-MTQ structures. Static light scattering experiments demonstrated that the BILBO1-NTD and the FPC4-CTD form a stable binary complex [ 14 ]. Using a combination of biophysical and cell biology approaches, we have shown that FPC4 binds to the horseshoe-like aromatic patch of the BILBO1-NTD [ 15 ].

The reported high-resolution NMR and crystal structures of the BILBO1-NTD demonstrate that it unexpectedly adopts a ubiquitin-like fold [ 13 , 14 ]. The C-terminal tail of the NTD is well-folded and rigidly wraps around the distal end of the elongated core structure. This tail helps to form a well-defined horseshoe-like pocket that harbours multiple highly conserved aromatic residues. On one side of the hydrophobic pocket, a gap is formed that leads to a pronounced negative trench at the bottom of the structure. Mutation of key residues within the pocket affect cell viability and impair the BILBO1 function in trypanosomes. Further, abolishing the Ca 2+ -binding ability of the EFh influences the shape and length of the polymers of BILBO1, disrupts the FPC structure, and affects trypanosome cell viability [ 9 , 10 , 15 ]. Deletion of both globular domains (i.e. NTD and EFh) leads to shorter polymers than those formed by the full-length BILBO1 [ 10 ], suggesting their role in facilitating inter-dimeric interactions.

A. The BILBO1 domains T3 (aa 171–587) and T4 (aa 251–587) were previously described in [ 9 ]. BILBO2 is presented as three domains: T1 (aa 1–103), T2 (aa 1–150), and the BILBO1-binding domain (B1BD, aa 151–271). FPC4 is presented as three domains: the microtubule binding domain (MT-BD), the coiled-coil domain (CCD), and the BILBO1 binding domain (B1BD). B. Y2H assay with full-length or domains of BILBO2 as bait and BILBO1 as prey, and of BILBO2 as prey and bait tested on minus histidine medium (-HIS). Loading control was on medium plus histidine. The positive control involved the previously demonstrated interaction between the p53 and T-antigen proteins, whereas the negative control involved Lamin and T-antigen proteins that do not interact.

BILBO1 is a modular protein with four structural domains [ 9 , 10 ] ( Fig 1A ). The globular N-terminal domain (NTD) is followed by two calcium-binding EF-hand motifs (EFhs), a central coiled-coil domain (CCD), and a C-terminal leucine zipper (LZ). The LZ is necessary but not sufficient for FPC targeting of BILBO1. The CCD allows the formation of filaments by the formation of antiparallel dimers that can extend into a polymer by the interdimer interaction between adjacent LZs. Indeed, BILBO1 was shown to form micrometre-long polymers and helical structures in vivo, in vitro and in a mammalian cell environment [ 4 , 9 – 12 ].

BILBO1 is the first identified FPC protein, with an indispensable role for the parasite [ 4 ]. RNA interference (RNAi) knockdown of BILBO1 in PCF cells prevents the biogenesis of a new FPC, a new FP, and a new FAZ, suggesting that the FPC is required for the biogenesis of numerous structures and their functions in the cell. In BILBO1 RNAi cells, the newly formed flagellum locates at the extended posterior end of the cell and is detached from the cell body. Furthermore, knockdown of BILBO1 is lethal in both PCF and BSF cells.

The shape of the trypanosome cell is maintained by a microtubule-based corset and by a flagellum laterally attached along the cell body. The flagellum is involved in cell mobility, kinetoplast segregation, and signal transduction [ 1 ]. It extends from the mature basal body (BB, tethered to the kinetoplast), and exits the cell through the FP. It then runs along the length of the cell while remaining attached to the cell body via the flagellum attachment zone (FAZ). Four specialized microtubules (the microtubule quartet, MTQ) nucleate at the BBs and extend around the FP, insert into the microtubule corset, and run as part of the cytoplasmic portion of the FAZ as far as to the anterior end of the cell body. The bulb-like FP is maintained by a ring-like cytoskeletal structure, the flagellar pocket collar (FPC), which encircles the neck of the FP around the exit site of flagellum beneath the cell surface [ 4 , 5 ]. The FPC is a complex structure, and in addition to its attachment to the flagellum, it is also attached to the microtubule cytoskeleton. Overlapping with the FPC is the hook complex (HC), a cytoskeleton-associated structure that is superimposed on top of the FPC throughout the cell cycle. The MTQ threads between these two structures [ 6 – 8 ].

During its life cycle, T. brucei is transmitted to the mammalian host via a blood meal of an infected tsetse fly. The parasite differentiates to several different forms in the insect and the mammalian host, among them the procyclic form (PCF) in the fly’s midgut, and the bloodstream form (BSF) in the mammalian bloodstream. Organelle positioning and segregation during the cell and parasite cycle show a high degree of coordination and control [ 3 ].

Trypanosomatids include many parasites of major medical and economic importance that cause several of the 20 World Health Organization’s listed neglected tropical diseases. These flagellated parasites share several unique features: a single mitochondrion with its compact genome (the kinetoplast, K), a flagellar pocket (FP), and a microtubule-based cytoskeleton to maintain cell shape and flagellar motility that plays crucial roles in life and cell cycle [ 1 ]. The FP is an invagination of the plasma membrane enclosing the base of the flagellum. In most trypanosomes, endo- and exocytosis occur exclusively through the FP. It thus provides the sole surface for numerous important receptors making them inaccessible for components of the innate immune system of the host. Moreover, the FP is responsible for sorting all parasite surface glycoproteins targeted to, or recycling from, the pellicular membrane and for removal of host antibodies from the cell surface. As such, the FP is a key player in protein trafficking, cell signalling and immune evasion [ 2 ]. Because it is hidden from the cell surface and sequesters important receptors, the FP is an attractive drug target. However, it has not been exploited as such because structural components of this organelle are still poorly characterized.

Results

BILBO2 localizes at the FPC primarily via its BILBO1-binding domain To determine the localization of BILBO2 in T. brucei, we generated a guinea pig BILBO2-specific antibody and cell lines expressing epitope-tagged fusion BILBO2 ( epitope BILBO2) using the pPOTv7 vector series for endogenous tagging [19] and controlled that the tag has no effect on cell growth (S2A Fig). Co-labelling of BILBO1 and BILBO2 on T. brucei detergent-extracted cells (cytoskeleton, CSK) revealed that BILBO2 and TY1 BILBO2 co-localized with BILBO1 at every stage of the cell cycle of PCF (Fig 3A and 3B) and of BSF (S2B Fig). To identify the FPC targeting domain of BILBO2, we generated PCF cell lines inducible for the ectopic expression BILBO2 HA and truncations BILBO2-T1 HA , BILBO2-T2 HA and BILBO2-B1BD HA . No dominant-negative phenotype or change in cell growth was observed after expression of any of these constructs (S3A Fig). Due to the ectopic expression system, BILBO2 HA was expressed at higher level than WT level as observed by western blot analysis, and a large pool was removed during extraction (S3B Fig). This pool was observed in the cytosol by IF and removed during detergent extraction (Fig 3C). A cytoplasmic pool was also observed with the BILBO2-T1 HA and BILBO2-T2 HA constructs, but to a lesser extent for BILBO2-B1BD HA that was less expressed (S3C Fig). Protein localization was assessed by immunofluorescence on WC and CSK, which showed that both BILBO2 HA and BILBO2-B1BD HA were detected at the FPC in extracted cells (Fig 3C). The BILBO2-T1 HA domain was removed during extraction, suggesting that it is not sufficient for FPC binding. Unlike for BILBO2-T1 HA , very weak but consistent BILBO2-T2 HA labelling was observed at the FPC (Fig 3C, asterisks in the main panels with increased contrast in the enlarged insets, and to be compared with BILBO2-T1 HA ), suggesting that the linker domain between the NTD and B1BD may be involved in FPC binding via another partner or that the T1 construct was too short for proper function. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Cellular localization of BILBO2. A. Immunolabelling on detergent-extracted PCF cells using anti-BILBO1 and anti-BILBO2 antibodies. B. Immunolabelling on detergent-extracted PCF cells expressing endogenously tagged TY1 BILBO2 using anti-BILBO1 and anti-TY1 antibodies. C. Ectopic expression of BILBO2 HA and domains was induced for 24H with 1 μg/mL of tetracycline followed by immunofluorescence on whole cells and detergent-extracted cells (Cytoskeleton). Unlike BILBO2-T1 HA , faint but consistent labelling of BILBO2-T2 HA the FPC on cytoskeleton is indicated by the asterisk and are to be compared in the insets with increased contrast. Scale bars in A, B and D represent 5μm, and 1 μm (insets). D. Sum intensity per collar quantification of BILBO1 and of TY1 BILBO2 labelling at the 1K1, 2K1N and 2K2N stages of the PCF cell cycle. Error bars represent the standard error (n = 200). https://doi.org/10.1371/journal.ppat.1009329.g003 Using DAPI as a marker for cell cycle stages (number of kinetoplasts and nuclei) and anti-BILBO1 and anti-TY1 tag, BILBO1 and TY1 BILBO2 levels were quantified on CSK PCF cells in four cell cycle stages using ImageJ (Fig 3D). The fluorescence intensity of BILBO1 remained constant at each FPC (old and new) during the cell cycle. Interestingly, the intensity of BILBO2 labelling varied dramatically during the cell cycle, with approximately 50% reduction in cells with 1 kinetoplast, 1 nucleus, but 2 FPCs (1K1N-2FPC, 2K1N-2FPC) in both the new and the old FPC. When the cell reached cytokinesis, the BILBO2 levels were almost equivalent to those at the beginning of the cell cycle. This suggests that BILBO2 expression is cell-cycle regulated in a different way to BILBO1. To further assess BILBO2 function in PCF and BSF of trypanosomes, we generated BILBO2 RNAi knockdown cell lines in the endogenously tagged background using the tetracycline-inducible RNAi system [20]. PCF and BSF cell growth were not affected after several days of induction, and no morphological phenotypes were observed despite the specific reduction of BILBO2 expression was observed by western blotting (S4 Fig) suggesting that BILBO2 may not be essential. However, several attempts to generate BILBO2 knock-out PCF and BSF cell lines failed. It is noteworthy that previous RNAi screen indicated that depletion of BILBO2 causes a deleterious effect on cell viability in both trypanosome life forms [21]. These data suggest that BILBO2 might play a critical role in the cell, whereas trace amount of proteins left in RNAi knockdown may have been sufficient to carry out such function and thus showed no defects in cell growth or other phenotypes.

Localization of BILBO2 to the FPC depends on BILBO1 We have reported previously that, upon RNAi knockdown of BILBO1, PCF cells display a cell cycle arrest in 2K2N stage and a detached new flagellum. Furthermore, the biogenesis of the new FAZ, FP, and FPC are also prevented, which eventually lead to cell death [4]. In addition, some of the FPC4 and MORN1 (a HC protein) proteins are mis-localized within the new detached flagellum [15]. Therefore, to analyse the fate of BILBO2 in the absence of a new FP and a new FPC, we generated an inducible BILBO1 RNAi cell line expressing TY1 BILBO2 (Fig 4). BILBO1 RNAi-induced cells stopped proliferating after 48h, and displayed a 2K2N growth arrest and detached new flagella (Fig 4A and 4B). Immunofluorescence on whole cells revealed that after 48h of induction BILBO2 was neither detected at the old flagellum nor at the new flagellum. Instead, a punctate cytosolic pool was observed (Fig 4B). Interestingly, western-blotting quantification showed that the CSK-associated pool of BILBO2 decreased by 5.3 x fold over the time-course of BILBO1 RNAi knockdown, while the total pool of BILBO2 (WC) increased by 2.6 x fold (Fig 4C and 4D). These data show that during BILBO1 knockdown, and thus in the absence of a new FPC, BILBO2 is still neo-synthetized but is no longer associated with the cytoskeleton. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Depletion of BILBO1 induces cytosolic localization of BILBO2. A. Comparative growth curves between WT cells and cells expressing TY1 BILBO2 and non-induced (NI) or induced (I) for BILBO1 RNAi. B. Immunolabelling of BILBO1 and TY1 BILBO2 on BILBO1 RNAi non-induced (NI) or induced 24H and 48H whole cells. C. Representative western-blot analysis of the fate of BILBO2 during BILBO1 RNAi in whole-cell (WC) and detergent-extracted samples (CSK). Anti-enolase and anti-tubulin were used as detergent extraction and loading controls, respectively. D. Quantification of the Western-blot in C. Error bars represent the standard error from two independent experiments. Scale bars in B represent 5 μm. https://doi.org/10.1371/journal.ppat.1009329.g004

The N-terminal domain of BILBO2 is homologous to the BILBO1 N-terminal domain BILBO1 is a multi-domain protein and its NTD interacts with FPC4-CTD via key residues in a conserved surface patch that are involved in BILBO1 function [15]. Database mining for proteins sharing a domain homologous to BILBO1-NTD identified the N-terminal domain of BILBO2. Alignment of BILBO1 and BILBO2 sequences revealed an overall similarity of 19% between the full-length proteins. However, the identity and similarity reach 32% and 38% respectively between their NTD (Fig 5A). Importantly, the two residues (Y64, W71), which were previously shown to play a critical role in BILBO1’s interaction with FPC4 and cell viability [9,13–15], are conserved or identical in BILBO2 (i.e. F63, W70). However, apart from its NTD and B1BD, no other structural or functional domains were identified in BILBO2. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. BILBO1 and BILBO2 share a conserved N-terminal domain with conserved residues. A. Alignment of the BILBO1 and BILBO2 NTD domains. Asterisks indicate identical residues; colons indicate conserved substitution; periods indicate semi-conserved substitutions. B. Immunolocalization of chimeric BILBO1-BILBO2 proteins. Anti-BILBO1 labels both BILBO1 and Ch BILBO1-BILBO2 HA ; anti-HA labels Ch BILBO1-BILBO2 HA or Ch BILBO2-BILBO1 HA . Cells were induced 18h with 1 μg.mL-1 tetracycline and detergent-extracted for immuno-labelling. Scale bars represent 5 μm. C. Cells inducible for BILBO1 RNAi and for the expression of recoded rec BILBO1 HA or rec BILBO2-BILBO1 HA were induced with 2 μg.mL-1 of tetracycline. The parental cells are inducible for BILBO1 RNAi only. Top panel: Western blot analysis of whole cells non induced or induced at different time points. Anti-TbSAXO was used as loading control. Bottom panel: Growth curves of non-induced and induced cells showing that rec BILBO1 HA can rescue the RNAi growth defect, contrary to rec BILBO2-BILBO1 HA . https://doi.org/10.1371/journal.ppat.1009329.g005 We hypothesized that if the NTD of BILBO1 and BILBO2 are functionally similar, they could be exchanged without affecting the function of either protein in vivo. Recoded chimeric BILBO1 and BILBO2 proteins with exchanged NTDs, namely Ch BILBO1-BILBO2 HA (BILBO1 aa1-118 fused to BILBO2 aa111-271) and Ch BILBO2-BILBO1 HA (BILBO2 aa1-110 fused to BILBO1 aa119-587), were ectopically expressed in PCF T. brucei (Fig 5B). It is important to note that, as previously described [9], ectopic long-term and/or high level expression of WT BILBO1, T3 or T4 was lethal due to excessive polymer formation induced by the CCD and LZ domains [9]. This explains the growth phenotype that occurs when expression of BILBO1 HA and of Ch BILBO2-BILBO1 HA is induced (S5 Fig). Nevertheless, using an anti-BILBO1 antibody recognizing aa1-110 of both BILBO1 and the chimeric Ch BILBO1-BILBO2 HA proteins, and an anti-HA antibody recognizing the chimeric proteins only, we immuno-localized endogenous BILBO1 and both chimeric proteins in detergent-extracted cells. Both Ch BILBO2-BILBO1 HA and Ch BILBO1-BILBO2 HA targeted to the FPC and, apart from the CCD-related problem mentioned above, no specific cell growth or morphology phenotype was observed (Figs 5B and S5). This supports the hypothesis that the NTDs are not involved in cellular localization but that they could have a similar functional role. The recoded BILBO1 HA and Ch BILBO2-BILBO1 HA constructs (i.e. resistant to the BILBO1 RNA interference) were also expressed in the BILBO1 RNAi background and were still expressed after BILBO1 RNAi induction (Fig 5C, western blot panel). However, whilst expression of recoded BILBO1 HA could rescue the BILBO1 RNAi growth phenotype (Fig 5C, growth curve panel), recoded Ch BILBO2-BILBO1 HA could not, and cells stopped growing in a similar time frame as the cells induced for BILBO1 RNAi only (Parental cells). This suggests that the NTDs are similar but are not identical domains that are not interchangeable in vivo.

BILBO2 is an FPC protein as well as a FPC4 interacting partner and binds to FPC4 via its N-terminal domain We previously demonstrated that FPC4 binds to microtubules via its N-terminus, and interacts with BILBO1 via its C-terminal region (FPC4-B1BD), suggesting a role in linking the FPC-HC-MTQ structure. Using a combination of site-directed mutagenesis, biophysical and cell biology approaches, we also showed that FPC4 binds to a conserved hydrophobic patch on the BILBO1-NTD surface patch. Further, static light scattering experiments demonstrated that the BILBO1-NTD and the FPC4-B1BD form a stable binary complex [15]. The recent high-resolution crystal structure of BILBO1-NTD and mutagenesis studies revealed that FPC4 interacts with BILBO1 by mainly contacting three aromatic residues W71, Y87, and F89 in the centre of the conserved hydrophobic patch [14]. Based on the high homology between NTDs of BILBO1 and BILBO2, we checked whether BILBO2-NTD also forms a stable complex with FPC4-B1BD. We first tested the interaction between BILBO2 and FPC4 (full-length or truncations) by Y2H (Fig 6A). Their interaction was confirmed with both full-length sequences. Further, BILBO2-NTD and FPC4-B1BD domains are both necessary and sufficient for the interaction. This was also supported by the proximity of FPC4 and BILBO2 in T. brucei immuno-fluorescence labelling at different stages of the cell cycle (Fig 6B), as it was observed for BILBO1 and FPC4 [15]. Because triple labelling of BILBO1, BILBO2 and FPC4 was challenging, probably due to primary and secondary antibodies steric hindrance, we turned to ultrastructure expansion microscopy (U-ExM) that allows the expansion of a sample and the visualization of preserved ultrastructure of macromolecules by optical microscopy [22,23]. This approach facilitated the localization of the FPC (labelled with BILBO1) in respect to the MTQ and to the axoneme that goes through (S6A Fig). The MTQ extends from between the mature and the immature basal bodies, turns around the FP and is prolonged along the FAZ as previously described in [5,6]. Higher resolution of the BILBO1 and BILBO2 colocalization was further determined using the same approach coupled to confocal microscopy (S6B Fig and S1 Movie). These approaches allowed us to evidence the annular shape of the FPC and the overall co-localization of BILBO1 and BILBO2 at the FPC. Further, triple labelling of BILBO1, BILBO2 and FPC4 showed that FPC4 partially colocalizes at the FPC and extends on the shank of the Hook Complex with a regular pattern as previously demonstrated in [15] (Fig 6C). Interestingly, BILBO2 colocalizes with BILBO1 at the FPC following also a regular pattern that extends past the FPC where it colocalizes with FPC4. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Interaction between BILBO2 and FPC4 is similar to that of BILBO1 and FPC4. A. Y2H interaction assay between BILBO2 and FPC4 and domains. B. Immuno-colocalisation of TY1 FPC4 and BILBO2 in detergent-extracted PCF cells. Scale bars 5 μm. C. Immunolocalization of BILBO1, myc BILBO2 and TY1 FPC4 using U-ExM in detergent-extracted PCF cells. Scale bars 10 μm and 5 μm in enlarged inset. D. Expression in U-2 OS cell and immunolocalization of FPC4 GFP and FPC4 deleted of its B1BD (FPC4-ΔB1BD GFP ) and HA BILBO2 and domains demonstrating that the BILBO2-T1 (aa 1–103) domain is not sufficient for a stable interaction between BILBO2 and FPC4 whereas a longer domain (BILBO2-T2) is stabilizing the interaction. Cells were detergent-extracted before the IF to reduce the FPC4 and BILBO2 cytosolic labelling. Scale bars 10 μm. E. A tripartite interaction is demonstrated in U-2 OS cells by the co-labelling of FPC4 and BILBO2 and domains onto the BILBO1 polymers. Scale bars 10 μm. F. Schematic representation of the interactions between BILBO1, BILBO2 and FPC4. https://doi.org/10.1371/journal.ppat.1009329.g006 We then took advantage of the property of FPC4 to bind to MT [15] to assess the localization of HA BILBO2 in detergent-extracted U-2 OS cells expressing FPC4 GFP (Fig 6D). When co-expressed, BILBO2 localized onto the MT labelling of FPC4, confirming their specific interaction in vivo. The deletion of the FPC4-B1BD abolished the interaction and resulted in the removal of BILBO2 during detergent extraction. Interestingly, BILBO2-T2 (aa1-151) can bind to FPC4, whereas BILBO2-T1 (aa 1–103) was extracted, suggesting that this construct might be too short for correct folding or disrupts the binding site for FPC4. Finally, the BILBO2-B1BD does not bind to FPC4 and is removed during extraction. Because BILBO2 binds to BILBO1 (via its CTD) and to FPC4 (via its NTD), we tested whether a tripartite interaction could occur in vivo (Fig 6E). Immuno-labelling of co-expressed BILBO1, HA BILBO2, and FPC4 TY1 in U-2 OS cells demonstrated that both FPC4 and BILBO2 can bind to the BILBO1 polymers (Fig 6E, a). This triple co-localization was also observed when BILBO2 deleted for its BILBO1 binding domain (BILBO2-T2, which binds to FPC4 but not to BILBO1) was expressed (Fig 6E, b) suggesting a tripartite interaction schematized in Fig 6F.

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