The unusual flagellar targeting mechanism and functions of the

© 2018. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

The unusual flagellar targeting mechanism and functions of the trypanosome orthologue of the ciliary GTPase Arl13b Yiliu Zhang1,2, Yameng Huang1, Amrita Srivathsan1, Teck Kwang Lim1, Qingsong Lin1 and Cynthia Y. He1,* 1

Department of Biological Sciences, Faculty of Science, National University of

Singapore, 14 Science Drive 4, Singapore 117543 2

Current address: Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive,

Singapore 138673 *

Corresponding author: [email protected]

Keywords: Arl13b; Arl3; Dimerization/Docking domain; flagellum; Trypanosoma brucei Summary The small GTPase Arl13b is one of the most conserved and ancient ciliary proteins. In

acts as a Guanine-nucleotide Exchange Factor (GEF) for Arl3 and is implicated in a variety of ciliary and cellular functions. We have identified and characterized TbArl13, the sole Arl13b homologue in the evolutionarily divergent, protozoan parasite Trypanosoma brucei. TbArl13 has conserved flagellar functions and exhibits catalytic activity towards two different TbArl3 homologues. However, TbArl13 is distinctly associated with the axoneme through a dimerization/docking (D/D) domain. Replacing the D/D domain with a flagellar membrane protein created a viable alternative to the wild-type TbArl13 in our RNA interference (RNAi)-based rescue assay. Therefore, flagellar enrichment is crucial for TbArl13 but mechanisms to achieve this could be flexible. Our findings thus extended the understanding of Arl13b and Arl13b-Arl3 pathway in a divergent flagellate of medical importance.

JCS Advance Online Article. Posted on 10 August 2018

Journal of Cell Science • Accepted manuscript

human and animals, Arl13b is primarily associated with the ciliary membrane, where it

Summary statement All roads lead to cilia: how trypanosome cell targets Arl13b to their flagellum in an unusual way

Introduction Flagella/cilia provide critical motility and sensory functions in eukaryotic cells from protists to mammals alike. Their roles in human physiology and diseases (ciliopathies) have spurred renewed interest in this ancient organelle (Badano et al., 2006; Hildebrandt et al., 2011). The cilium is topologically an open organelle (not fully enclosed by membrane), and relies on elaborate protein trafficking/gating systems to maintain its molecular identity and functions (Garcia-Gonzalo and Reiter, 2012; Ishikawa and Marshall, 2011). The small GTPase Arl13b localizes predominantly to the ciliary membrane in all ciliated model systems studied thus far and has been widely adopted as a ciliary marker (Cantagrel et al., 2008; Cevik et al., 2010). Studies have shown that Arl13b targeting involves multiple mechanisms, requiring at least two sequence elements essential for localization: an N-terminal palmitoylation motif that facilitates membrane association, and a less conserved VxP motif containing C-terminus tail that restricts the protein to the ciliary membrane. However, these elements are not exhaustive and other

2008). The ciliary localization of Arl13b is believed to be critical for its functions, as Arl13b transgenes that contain targeting-related mutations fail to rescue Arl13b loss-offunction phenotypes (Duldulao et al., 2009; Higginbotham et al., 2012). Mutations in Arl13b are associated with Joubert Syndrome (a ciliopathy), and Arl13b null animals display a wide range of ciliary defects that compromise ciliogenesis, cilia length extension, ciliary motility and sonic hedgehog signaling (Caspary et al., 2007; Cevik et al., 2010; Duldulao et al., 2009; Larkins et al., 2011; Li et al., 2010; Lu et al., 2015; Sun et al., 2004). Concomitant with these pleiotropic phenotypes, various molecular mechanisms have been ascribed to Arl13b, including the regulation of IFT (Cevik et al., 2010; Li et al., 2010; Nozaki et al., 2017), cooperation with the exocyst


factors might also be at play (Cevik et al., 2013; Higginbotham et al., 2012; Hori et al.,

(Seixas et al., 2015), and direct recruitment of other important ciliary proteins such as INPP5E (Humbert et al., 2012). The best studied and most conserved role of Arl13b discovered so far is being a GEF for Arl3 (Cuvillier et al., 2000a; Gotthardt et al., 2015; Hanke-Gogokhia et al., 2016; Ivanova et al., 2017; Schrick et al., 2006; Zhang et al., 2016) - another Arf/Arl family member implicated in ciliary functions (Cuvillier et al., 2000a; Gotthardt et al., 2015; Hanke-Gogokhia et al., 2016; Ivanova et al., 2017; Schrick et al., 2006; Zhang et al., 2016). It has been proposed that the exclusive ciliary localization of Arl13b (Arl3-GEF) creates an Arl3-GTP gradient between the cilia and the cytoplasm, which in turn facilitates the directional transport of ciliary proteins. However, this model only applies to lipidated ciliary proteins, which may not readily explain the wide range of ciliary defects seen in Arl13b and Arl3 mutants. Arl13b is believed to be one of the most ancient ciliary proteins (Sung and Leroux, 2013), yet little is known regarding its role outside the metazoan systems. We serendipitously discovered that in the uni-flagellated protozoan parasite Trypanosoma brucei, the single homologue of Arl13b (TbArl13) lacks both the palmitoylation motif and the C-terminus tail – both essential for Arl13b ciliary targeting in higher eukaryotes. In light of recent evidence that Arl13b may also have roles outside of the cilia (Casalou et al., 2014; Mariani et al., 2016), we originally hypothesized that TbArl13 might represent an evolutionarily-divergent, non-ciliary form of Arl13b. However, we found that TbArl13 is targeted to the flagellum by associating with the axoneme, through a domain

regulatory subunit in mammalian cells. Replacing the D/D domain in TbArl13 with calflagin Tb24, a trypanosome flagellar membrane associated protein, enriches the chimera protein at the flagellar membrane. Interestingly, this chimera TbArl13 mutant could functionally rescue cell death phenotypes upon depletion of endogenous TbArl13, suggesting that the ‘end’ of flagellar enrichment is more critical than the ‘means’ of targeting. Finally, we showed that the Arl13b-Arl3 pathway in T. brucei is conserved but bifurcated, in that TbArl13 catalyzes two differentially localized, functionally important Arl3 homologues.


similar to the Dimerization/Docking (D/D) domain of the PKA (protein kinase A)

Results TbArl13 is an Arl13b orthologue with unusual features. Reciprocal BLAST analyses have identified Tb927.10.5230 as a homologue of human Arl13b. Both E-values and identity/similarity scores are within the range reported for other published Arl13b homologues ((Cevik et al., 2010) and Fig. S1A). To further analyze Tb927.10.5230, we performed phylogenetic reconstruction, sampling all predicted trypanosome Arf/Arl GTPases and major Arf/Arl family members from representative eukaryotic model systems (Fig. S1B). The resulting phylogenetic tree confidently placed Tb927.10.5230 in the same clade with all other known Arl13/Arl13a/Arl13b proteins included in the dataset (Fig. S1B). Because the duplication of Arl13 to Arl13a and Arl13b is likely a vertebrate invention (Kahn et al., 2014), we have named the trypanosome orthologue TbArl13 to reflect its homology to both Arl13a and Arl13b. Due the scarcity of functional information on Arl13a, we limit our discussion to be in the context of Arl13b in this work. Multiple sequence alignment and domain comparison of Arl13b proteins (Fig. 1A and Fig. S2) show that TbArl13 possesses a highly conserved GTPase domain, with characteristic absence of the ‘Switch 2’ catalytic glutamine (DXXGQ) that is highly conserved in other small GTPases (Cevik et al., 2010; Miertzschke et al., 2014). Immediately following the GTPase domain is a predicted coil-coil motif (CC), which is

Miertzschke et al., 2014). Notably, TbArl13 does not have the N-terminal palmitoylation site found in most other Arl13b members except for CrArl13b (Fig. S2). TbArl13 also lacks a long C-terminal tail – another characteristic albeit less conserved feature of Arl13b (Li et al., 2010). The most unusual feature of TbArl13 is the presence of an N-terminal extension, which is predicted to be a ‘Dimerization/Docking domain of the PKA regulatory subunit’ (D/D, NCBI conserved domain ID: cl02594; Superfamily ID SSF47391; Interpro ID: ipr003117). D/D is a class of protein targeting domains best characterized in PKA but have also been found in non-PKA proteins (Banky et al., 1998; Carr et al., 2001; Sivadas et al., 2012). The D/D domain is not present in Arl13b proteins of higher eukaryotes but


also unique to Arl13b proteins and required for their catalytic function (Hori et al., 2008;

is highly conserved among Arl13b homologues in the kinetoplastid group of protozoans (Fig. S3). Together, our bioinformatic analyses show that TbArl13 has highly conserved catalytic domains (GTPase domain and the CC motif) but is divergent in elements that have been implicated in targeting and/or regulatory functions.

TbArl13 localizes to the flagellum and associates with the axoneme. We first investigated the intracellular localization of TbArl13 in PCF T. brucei cells (the proliferative form found in tse-tse fly midgut) by tagging one endogenous TbArl13 allele with a yellow fluorescent protein (YFP) reporter followed with a triple HA tag (Fig. 1B). Highly specific polyclonal antibodies were also raised against TbArl13 (Fig. 1C). In all three approaches, TbArl13 displayed flagellar localization with stronger signal intensity towards the proximal end (Fig. 1B-G). Some weak cytoplasmic signals were also observed in PFA-fixed whole cells (Fig 1B, top row). TbArl13 association with the flagellum was at least partially resistant to detergent extraction (Fig. 1B, bottom row), suggesting association with the flagellar cytoskeleton. Similar expression and localization of TbArl13 was also found in the mammal-infectious bloodstream form (BSF) cells (Fig. S4 A and B). Using a protocol that efficiently extracts membrane associated proteins such as the dual acylated calflagin Tb24 (Tyler et al., 2009), we consistently observed a cytoskeletal pool for TbArl13 (Fig. S4C).

axoneme is nucleated from the basal bodies, the paraflagellar rod (PFR) only assembles after the flagellum exits the flagellar pocket and at a distance to the basal bodies ((Bastin et al., 2000; Kohl et al., 1999), and Fig. 1G). TbArl13 signal began at precisely the same position as the axoneme marker YFP-RSP3 (Diener et al., 1993; Ralston et al., 2006), with the two signals overlapping completely until TbArl13 intensity diminished towards the distal end (Fig. 1D). The gradient-like distribution of TbArl13 along the axoneme was further illustrated with intensity plotting of both anti-TbArl13 and radial spoke protein 3 (YFP-RSP3) (Fig. 1E). TbArl13 however, did not colocalize with the PFR and a gap was evident between the proximal ends of PFR and TbArl13 (Fig.1F, green and red arrows). TbArl13 was also readily observed in nascent flagella where no PFR had assembled


There are two major cytoskeletal structures in the trypanosome flagellum: while the

(Fig.1F, white arrowhead). Based on the above observations, we conclude that TbArl13 localizes to the flagellum and associates with the axoneme.

The D/D domain is required for flagellar targeting and axonemal association of TbArl13. The D/D domain was first characterized and best studied in the regulatory subunits of PKA (Lester and Scott, 1997; Rubin, 1994). The subcellular localizations of the PKA regulatory subunits are regulated through the docking of its D/D domain to AKAPs (Akinase Associated Protein) found in various intracellular locations. To investigate if the D/D domain in TbArl13 plays a role in its flagellar targeting, we generated stable cell lines containing tetracycline-inducible expression of YFP fused to full length (FL-YFP) and truncated TbArl13 (DD-YFP and CD-YFP). DD-YFP contained the D/D domain (162 aa), whereas CD-YFP spanned the GTPase domain and the CC motif (61-270 aa). All three recombinant proteins were found throughout the PFA-fixed cells possibly due to overexpression, but only FL-YFP and DD-YFP demonstrated a clear enrichment at the flagellum, which was more obvious upon detergent extractions (Fig. 2A). In contrast, CD-YFP signal was completely lost in detergent-extracted cells. Cell fractionation analyses also confirmed that both FL-YFP and DD-YFP had a detergent-resistant pool, while CD-YFP was completely soluble (Fig. S5A). These results demonstrate that the D/D domain fully accounts for the flagellum targeting and axonemal association of

Since the D/D domain is also known to mediate PKA regulatory subunit dimerization (Scott et al., 1990), we asked whether TbArl13 can self-associate. Coimmunoprecipitation (Co-IP) assays showed that endogenous TbArl13 co-precipitated with both FL-YFP and DD-YFP but not with CD-YFP or YFP alone (Fig. 2B). These results suggest that TbArl13 self-associates through the D/D domain, and that the D/D domain of TbArl13 likely functions in the similar ways as the Dimerization/Docking domain of PKA.


TbAr113.

If TbArl13 solely relies on the D/D-mediated axoneme docking for its flagellar targeting, inhibition of the docking process would not only disrupt the axoneme association but also its overall flagellar enrichment. We reasoned that excess amounts of D/D domains alone might compete with TbArl13 for docking sites, serving as an inhibitor of TbArl13 docking. To test this hypothesis, we overexpressed D/D domain (mCherry-DD) using a cumate (Cmt)-inducible system(Li et al., 2017) in cells expressing endogenously tagged TbArl13-mNeonGreen. In detergent-extracted cells, we consistently observed weaker flagellar mNeonGreen signals in detergent-extracted cells, we consistently observed weaker flagellar mNeonGreen signals (Fig. 2C, quantifications shown in Figs. 2E and S5B) and reduced TbArl13-mNeonGreen in the cytoskeletal fraction (Fig. 2F) in cells expressing mCherry-DD. Remarkably, overall flagellar enrichment of TbArl13mNeonGreen in live cells was also reduced (Fig. 2D). Similar results were obtained using 3xHA-DD, thereby ruling out possible fluorescence energy transfer effects between mNeonGreen and mCherry (data not shown). These results therefore support a diffusionretention model for TbAr113 flagellar targeting, where TbArl13 enters the flagellum by diffusion but is retained in the flagellum by D/D mediated axoneme docking (see Fig. 7). Furthermore, the number of TbArl13-D/D docking sites on the axoneme is likely limited. Notably, overexpression of the TbArl13 D/D domain is not well tolerated by the cells as high expression levels could not be maintained for more than 2 days (data not shown). The dominant negative effect of D/D overexpression will be discussed later in BSF cells

TbArl13 is essential for flagellum biogenesis and cell survival. The cellular functions of TbArl13 were investigated by Arl13 inducible knockout (iKO) in PCF cells. Both endogenous TbArl13 alleles were replaced with drug resistance genes, and ectopic TbArl13-YFP expression was achieved through a tetracycline (Tet) inducible cassette stably incorporated into the genome (Fig. S6A). 72hrs after tetracycline washout, TbArl13 expression was not detectable (Fig. S6B). Unlike control cells that each contains single or duplicated flagella that tightly adhere to the cell body via the flagellum attachment zone (FAZ), TbArl13-iKO cells displayed severe flagellar defects (Fig. 3A


where it is more pronounced.

and B, arrows). The flagella (marked by anti-PFR) became significantly shorter (from 18.7 ± 0.25 μm in control cells to 13.2 ± 0.44 μm in iKO cells, 72hours post induction). FAZ (marked by L3B2) length also reduced dramatically (from 14.5 ± 0.35 μm in control cells to 6. 8 ± 0.54 μm in iKO cells, 72hours post induction), with some L3B2 signals appearing as condensed stubs at 72hrs post iKO induction (Fig. 3A and C, arrowheads). Both flagella and the associated FAZ are key regulators in trypanosome cytokinesis (Kohl et al., 2003; Robinson et al., 1995; Vaughan et al., 2008; Zhou et al., 2011), this may explain the appearance of multi-nuclear, multi-flagellated ‘monster cells’ in TbArl13-iKO population (Fig. 3A, asterisks). Consistently, TbArl13 iKO cells could not proliferate beyond 72 hours (Fig. 3D). To find out whether the shortening of flagella was associated with ultrastructural defects, TbArl13 iKO cells were examined using transmission electron microscopy (TEM) (Fig.4E). For flagella (or regions of flagella) that were still attached to the cell body, the associated FAZ filament appeared much wider in iKO cells than in WT cells (Fig. 3E, brackets). This observation is consistent with the condensed appearance of FAZ stubs marked by L3B2 staining (c.f. Fig. 3A). Strikingly, flagella with severe ultrastructural defects could be seen in the iKO cells but never in control (Fig. 3E). The defects were heterogenous - both axonemes and PFR could be malformed, with the axonemal defects ranging from misplaced microtubule doublets to total disruption of the 9+2 pattern. Occasionally, two axonemes could be observed in the same flagellar lumen. These wide-

flagellum biogenesis. We also studied TbArl13 knockdown phenotypes in the BSF cells. TbArl13-RNAi completely arrested BSF cell proliferation within 48hrs of induction (Figs. 3F and S6C), generating ‘monster cells’ with multiple flagella and nuclei, indicative of cytokinesis failure (Fig. 3G and H). Despite occasional flagellum detachment and FAZ shortening (Fig. 3G arrowhead and asterisk), we did not observe any major change to flagellar length, nor gross perturbation in flagellar ultrastructure as seen previously in the PCF cells. It has long been noticed that cytokinesis and cell survival of BSF cells are much more sensitive to flagellum perturbation than in PCF (Broadhead et al., 2006; Ralston and


ranging phenotypes suggest that the loss of TbArl13 results in total derangement of

Hill, 2006; Wheeler et al., 2013). The acute cytokinesis arrest and cell death in BSF following TbArl13 knock down may have prevented the progressive manifestation of flagellar defects as seen in PCF cells. Together, these results indicate that TbArl13 is an essential protein in T. brucei and is a critical player in flagellum biogenesis.

Flagellar enrichment of TbArl13 is crucial for its cellular functions. We next investigated whether the function of TbArl13 depends on its distinct flagellar targeting. To this end, we devised a double-inducible RNAi complementation assay in BSF cells. In this assay, Cmt-inducible expression vectors containing different, RNAiresistant (iR) TbArl13 mutants were stably transfected into BSF cells with stable integration of Tet-inducible TbArl13-RNAi (Fig. 4A and B). As shown in Fig. 4 C, the expression of full-length, RNAi-resistant TbArl13 (FLiR-YFP) was able to restore cell proliferation when native TbArl13 was depleted via RNAi, thereby validating the experiment setup and confirming the specificity of TbArl13-RNAi phenotypes described earlier. Expression of the R133Q mutant (RQiR-YFP), which corresponds to the disease associated R79Q mutation in human Arl13b, could not rescue TbArl13-RNAi. The coil-coil domain, which has been shown to be involved in the GEF activity of Arl13b (Gotthardt et al., 2015), is also indispensable as CCtiR-YFP failed to rescue the growth of TbArl13 RNAi cells. Consistent with results observed in PCF cells

the axoneme and could not rescue TbArl13-RNAi in BSF. These results suggest that D/D mediated flagellar targeting is required for the cellular functions of TbArl13. It remained unclear, however, whether D/D had any functional role beyond flagellar targeting. Additionally, it was not clear whether flagellar enrichment and axoneme association are both required for TbArl13 functions, since D/D deletion abolished both attributes. To address these issues, we generated a chimeric protein (Tb24CDiR) by swapping the D/D domain of TbArl13 with the well-established flagellar membraneassociated protein calflagin Tb24 (Tyler et al., 2009). As expected, Tb24CDiR-YFP exhibited a flagellar membrane localization and could be extracted by detergent, both


(c.f. Fig. 2A), CDiR-YFP did not show enrichment in the flagellum nor association with

features distinct to wild type TbArl13 (Fig. 4D and Fig. S6D). Strikingly, Tb24CDiRYFP was able to support continued cell proliferation, in the absence of endogenous TbArl13 (Fig. 4C and E). These results indicate that the flagellar localization of the catalytic domain of TbArl13 (GTPase domain and CC motif) is required to support BSF cell proliferation. However, the exact location of the protein, membrane or cytoskeletonassociated, and the mechanisms through which flagellar enrichment is achieved could be flexible. Interesting still, we found that DDiR-YFP expression alone had an inhibitory effect on cell proliferation before expression was lost and growth recovered (Fig. 4C, blue asterisk). Microscopy data showed rapid accumulation of ‘monster cells’ at a higher induction level (15 µg/ml instead of 2 µg/ml) (Fig. 4F). Combined with our earlier finding that DD-YFP overexpression displaces endogenous TbArl13 (Fig. 2C-F), this finding lends further support to our conclusion that flagellar enrichment of TbArl13 is critical in T. brucei.

TbArl13 catalyses both TbArl3A and TbArl3C To investigate the interaction network of TbArl13, we performed proximity-dependent biotin identification (BioID) of potential TbArl13 interacting partners (see materials and methods for details). Specific protein hits were ranked based on their relative protein 4th and 7th most abundant proteins identified through BioID. Further, a protein (Tb927.10.5810) homologous to a known Arl3 effector BARTL1 (Lokaj et al., 2015) was also found. The BioID results suggest that The Arl13b-Arl3 pathway is likely to be conserved in T. brucei. Three Arl3 homologues have been predicted in T. brucei (Cuvillier et al., 2000b; Price et al., 2005) (Fig. S2). The localization of all three Arl3 homologues were first screened by overexpressing YFP-tagged recombinant proteins. TbArl3A-YFP was enriched at the nucleus and along the flagellum, as well as forming a punctum at the base of the flagellum (Fig. S7A). TbArl3C-YFP was highly concentrated at the base of the flagellum,


content (Table 1). Notably, two Arl3 homologues named TbArl3C and TbArl3A were the

with a weaker presence along the flagellum. TbArl3B-YFP, however, appeared to be completely excluded from the flagellum, and adopted a reticulated pattern that closely resembled the PCF mitochondrion (Povelones et al., 2013). The differential localization of TbArl3A and TbArl3C was further validated by endogenously tagging both Arl3 genes in the same cell line for direct comparison (Fig. 5A). While TagRFPt-TbArl3C localized almost exclusively at the basal bodies, TbArl3AmNeonGreen was additionally present and enriched along the flagellum. The nuclear localization of TbArl3A-YFP seen under overexpression conditions was not evident in cells with endogenously expressed TbArl3A-mNeonGreen, and therefore could be due to overexpression. Bi-fluorescence complementation (BiFC, (Kodama and Hu, 2010b) was employed to study the interaction between TbArl13 and TbArl3 variants in vivo (Fig. S7B). Consistent with the BioID results, only TbArl3A-VC and TbArl3C-VC exhibited positive interaction with TbArl13-VN, allowing recombined Venus fluorescence to be detected both at the base and along the flagellum (Fig. 5B). Additionally, bacterially produced His-TbArl13 could pull-down TbArl3A-VC and TbArl3C-VC, but not TbArl3B-VC from T. brucei cell lysates (Fig. 5C). Reciprocal in vitro pull-down experiments also confirmed direct interactions of His-TbArl13 with GST-TbArl3A and GST-TbArl3C, respectively (Fig. S7C and D).

TbArl3C could be regulated by TbArl13 in the same fashion, we performed GEF assays using fluorescent, mant-labeled nucleotides (Gotthardt et al., 2015; Zhang et al., 2016). His-TbArl13 greatly accelerated mantGDP dissociation from both TbArl3A and TbArl3C, in the presence of excess unlabeled GTP (Fig. 5D-G). His-TbArl13 also accelerated the association of TbArl3A and TbArl3C to mantGTP (Fig. S7E and F). Interestingly, TbArl3C seems to have a much higher intrinsic nucleotide exchange rate compared to TbArl3A, further suggesting that the two Arl3 homologues may be regulated and/or function differentially. Bacterially produced GST-TbArl3B was not soluble in our hands. Considering that TbArl3B did not interact with TbArl13 in BiFC and in vivo pull-


Arl13b has been shown to be a GEF for Arl3. To investigate whether both TbArl3A and

down assays, it was unlikely an interacting partner of TbArl13 and was thus not included in the GEF activity assays.

Both TbArl3A and TbArl3C function in flagellum biogenesis. To investigate if TbArl3A and TbArl3C have flagellar functions, we generated classic GTP-locked mutants by altering the catalytic glutamine at Switch 2 region to leucine. Inducible expression of TbArl3A-Q70L-BB2 and TbArl3C-Q77L-BB2 both drastically inhibited flagellum biogenesis, with both flagellum and FAZ markers reduced into stubs or dots in most cells at 20hrs post induction (Fig. 6A and C). In stark contrast, expression TbArl3B-Q71L-BB2 had no observable impact on flagellum or cell morphology (Fig. 6B). In agreement with the flagellar defects, expression of TbArl3A-Q70L-BB2 or TbArl3C-Q77L-BB2 both inhibited cell proliferation. TbArl3B-Q71L-BB2 had no adverse impact on cell survival in culture (Fig. 6D-F), despite a higher expression level compared to the other two mutants (Fig. 6G). These results demonstrated that only TbArl3A and TbArl3C, both of which interact with TbArl13, are functionally relevant in flagellum biogenesis.

Discussion

Multiple lines of evidence support that TbArl13 is a bona fide Arl13b orthologue. At the sequence level, the catalytic domain is highly conserved among TbArl13 and other Arl13b orthologues. At the cellular level, TbArl13 localizes to the flagellum and is critical for flagellar biogenesis and functions. At the molecular mechanism level, TbArl13 catalyzes guanine nucleotide exchange in trypanosome Arl3 homologues. Trypanosomes (euglenozoan) are one of the most divergent groups of extant eukaryotes (Cavalier-Smith, 2010). These highly conserved attributes firmly support that the role of Arl13b and the Arl13b-Arl3 pathway in ciliogenesis was established early in evolution.


Here we reported a highly unusual Arl13b orthologue in the protozoan parasite T. brucei.

Unlike the predominant flagellar membrane localization of Arl13b in animal cells, TbArl13 associates with the axoneme, along which it forms a gradient-like pattern. Interestingly, ARL-13 in C. elegans is also highly enriched at the proximal segment in amphid and phasmid cilia, albeit on the membrane domain (Cevik et al., 2010; Cevik et al., 2013). The same studies also found proximal enrichment of mammalian Arl13b in the cilia of MDCKII, mouse oviduct and tracheal epithelial cells. It is striking that fundamentally different flagellar targeting mechanisms, which target different subflagellar domains, result in a similar Arl13b pattern along the flagellum/cilium. This appears to be a case of convergent evolution, where selective pressure favors strong Arl13b presence at the proximal end of cilia. A recent study suggests that after Arl3-GTP dependent release, ciliary cargoes (INPP5E) may be relayed onto the IFT machinery for trafficking within the cilium (Kosling et al., 2018). In this perspective, one advantage that the asymmetrical distribution of the Arl3-GEF could provide is the maximization of cargo release efficiency near the ciliary gate (where it is picked up by IFT). However, this advantage is likely not essential, as suggested by our rescue results and the observations that not all cell types show such proximal Arl13b enrichment at the cilia. We have shown that the axonemal association of TbArl13 is mediated by its single D/D domain, which is conserved within the kinetoplastid group but absent in other eukaryotes. D/D domains were first described and are best studied in the targeting of mammalian PKA. However, accumulating evidence (including this study) suggest that they are found

Fujita et al., 2000; Sivadas et al., 2012). Thus, D/D domains may represent a class of modular, generally adaptable domain for sub-cellular protein targeting. Based on the observation that overexpressed D/D domain not only displaced TbArl13-mNeonGreen from the axoneme, but also weakened the latter’s overall flagellar enrichment, we propose that a ‘diffusion-retention’ model can account for TbArl13’s flagellar targeting/enrichment (Fig. 7). In this model, TbArl13 could freely diffuse between the cytoplasm and flagellar lumen but is predominantly enriched in the flagellum due to its affinity with the axoneme. This affinity of TbArl13 to the axoneme is conferred by the D/D domain, likely through specific interaction with a putative axonemal docking partner. Indeed, while ciliary membrane protein entry is tightly regulated by a barrier


in a wide range of non-PKA proteins including some flagellar proteins (Carr et al., 2001;

system including the transition zone complex and a septin ring(Hu et al., 2010; Reiter et al., 2012), recent studies suggest that the ‘ciliary gate’ for soluble proteins is more ‘leaky’ and may grant ciliary access to proteins up to 100KD (Breslow et al., 2013)or even 650KD (Lin et al., 2013). Therefore, the diffusion-retention mechanism may play a significant role in the ciliary targeting of smaller soluble proteins (Nachury, 2011). Taking advantage of this simple D/D-mediated flagellar targeting mechanism, we explored the relationship between the localization and functions of TbArl13. The chimeric Tb24CDiR-YFP, which could enrich on the flagellum membrane but did not form a gradient distribution along the flagellum, could functionally replace WT TbArl13. On the other hand, CDiR-YFP, which could enter the flagellum but showed no enrichment compared to the cytoplasm, could not rescue TbArl13 RNAi cells. These results suggest that neither the axoneme association nor the gradient-like distribution is essential for Arl13 function. Instead, flagellar enrichment of the TbArl13 catalytic domain, via D/D domain or Tb24, is required (Fig. 7A-C). The flagellar enrichment of TbArl13 may be crucial for its function as an Arl3-GEF, generating a greater concentration of Arl3(A/C)-GTP inside of the cilia, thereby facilitating directional transport of ciliary cargoes (Fansa et al., 2016; Gotthardt et al., 2015; Ismail et al., 2011). A recent study showed that a myristoylated (instead of palmitoylated) Arl13b mutant can localize to the cilia but cannot fully complement the loss of endogenous Arl13b functions (Roy et al., 2017). We nevertheless noted that the myristoylated mutant outperformed all

defects both in Arl13b-null cells and fish. Additionally, the poor protein stability of this Arl13b mutant could have been a major factor preventing a full rescue. The dominant-negative effects of expressing just the D/D domain could be due to its activity as an orthosteric inhibitor, as these domain truncations can compete with WT TbArl13 for docking partners on the axoneme (Fig. 2 C-F and Fig. 7 D-F). Since D/Dmediated Arl13b targeting is not found in other eukaryotes except for the kinetoplastids, this docking machinery presents an attractive therapeutic target for a range of human and animal diseases caused by this group of parasites. Indeed, the D/D-AKAP interaction in the PKA pathway is known to be susceptible to interference with short peptides,


other non-ciliary-localized Arl13b mutants tested in rescuing the ciliary biogenesis

peptidomimetic or small molecules, and is being explored as a potential therapeutic target (Christian et al., 2011; Hundsrucker and Klussmann, 2008; Tröger et al., 2012; Wang et al., 2014). TbArl13 likely dimerizes due to the D/D domain. Interestingly, it was reported that human Arl13b (which does not possess a D/D domain) could also self-associate through the ‘N-terminal domain’ (Hori et al., 2008). A later biochemical study of CrArl13b provided strong evidence against Arl13b dimerization, but the recombinant protein used in the study appeared to be a truncation that lacks residues 1-17 (Miertzschke et al., 2014). If the extreme N-terminal residues in animal Arl13b indeed facilitate dimerization, Arl13b dimerization would merit further study. Regardless, Arl13b dimerization is likely not required for Arl3 GEF activity, since the truncated CrArl13b was used to first characterize the GEF activity (Gotthardt et al., 2015). Similarly, our positive rescue results with Tb24CDiR-YFP suggest that dimerization via D/D is not obligatory. It is possible that Tb24 might form dimers, though current evidence is inconclusive (Xu et al., 2012). Although Arl3 has not been found to be associated with any ciliopathies, classic ciliopathy manifestations have been reported in Arl3 knockout and conditional knockout mice (Hanke-Gogokhia et al., 2016; Schrick et al., 2006). The first evidence suggesting a ciliary function for Arl3 came from pioneering research done in the trypanosomes, where

in several species (Cuvillier et al., 2000b; Sahin et al., 2004). Here, we show that expression of GTP-locked forms of TbArl3A and TbArl3C both inhibited flagellum biogenesis. TbArl13 binds and exhibits GEF activity to both TbArl3A and TbArl3C, while TbArl3C has much higher intrinsic nucleotide exchange activity than TbArl3A. Interestingly, TbArl3A and TbArl3C adopt different localizations, with TbArl3A featured prominently along the flagellum and TbArl3C highly enriched on the basal body. The previously annotated TbArl3B (Cuvillier et al., 2000b; Price et al., 2005) did not interact with TbArl13 nor caused any observable phenotypes when its GTP-locked form was expressed. Taken together with the observation that it did not fall under the Arl3 clade in our phylogenic analysis (Fig. S1B), it is uncertain whether TbArl3B is a true Arl3


the expression of the GTP-locked form of Arl3A severely inhibited flagellum biogenesis

orthologue. Our results suggest a possible expansion and/or specialization of Arl3 functions in T. brucei. Considering the well-established role of Arl3 as a cargo displacement factor, an intriguing scenario is that the differential localization of the two TbArl3 proteins allows for different cargoes to be released at different flagellar locations. Mammalian cells have only one Arl3, which is found in multiple structures including the centrioles (or basal bodies of cilia), the cilia and the Golgi apparatus (Grayson et al., 2002; Zhou et al., 2006). It is possible that in higher eukaryotes, Arl3 cargoes are also location-dependent and this may help to explain its multiple roles during ciliogenesis (Hanke-Gogokhia et al., 2016). Further dissection of TbArl3A and TbArl3C functions could shed more light into the complex role of Arl3 in cilia biology.

Materials and Methods Bioinformatics Putative Arf/Arl GTPases were retrieved by searching ARF domain (Domain ID: IPR024156) in the Tritryp Database (http://tritrypdb.org) (Aslett et al., 2010). Large proteins(>40KD) were removed from the list. Sequences of Arf/Arl family members from other eukaryotic model systems were retrieved from NCBI GenBank (Benson et al., 2013). Phylogenetic analysis was carried out using both Maximum Likelihood (ML) and Bayesian phylogenetic inferences. Initial sequence alignment was carried out in mCoffee

downstream analyses. The GTPase domains were re-aligned using MAFFT-LiNSI (v 7.394) (Katoh and Standley, 2013). Alignment was next masked using ALISCORE (v 2.0) (Kück et al., 2010; Misof and Misof, 2009) and ALICUT (v 2.31) (https://github.com/PatrickKueck/AliCUT) to remove the positions that were not unambiguously aligned. Both Maximum Likelihood (ML) and Bayesian phylogenetic inferences were conducted on the resulting alignment. Model testing was done with ProtTest 3 (v3.4.2) (Darriba et al., 2011). The best fit model was selected based on Akaike Information Criterion (AIC). Maximum Likelihood inference was carried out using RaxML (v8.2.10) (Stamatakis, 2014), where the best scoring tree was obtained using twenty alternative runs on distinct starting trees. Multiparametric bootstrapping was


(Wallace et al., 2006), based on which the Arf/Arl GTPase domains were extracted for

carried with automatic bootstrapping option (autoMRE). Bayesian phylogenetic inference was conducted using MrBayes (v3.2.6) (Ronquist and Huelsenbeck, 2003). Two parallel analyses were run for 10 × 106 generations with four chains each. Sampling frequency was set at 1000 and initial 103 trees were discarded as burn-in. A consensus of the two trees was obtained and the convergence was assessed using Tracer (v1.6) (Rambaut et al., 2015). Identity and similarity scores were calculated and tabulated using the SIAS program (imed.med.ucm.es/Tools/sias.html). Multiple sequence alignments were visualized using Sequence Manipulation Suite (Stothard, 2000). D/D domain in TbArl13 was identified using NCBI Conserved Domains and Interpro (https://www.ebi.ac.uk/interpro/). COILS server was used for coiled-coil prediction (Lupas et al., 1991). Palmitoylation prediction was conducted using NBA-Palm (Xue et al., 2006).

Cell culture and transfection All PCF cells were maintained in Cunningham medium supplemented with 15% heatinactivated fetal bovine serum (Hyclone) at 28℃. All BSF cells were maintained in HMI9 medium containing 10% heat-inactivated fetal bovine serum (Hyclone) at 37℃ with 5% CO2. Cell transfections were carried out based on published protocols (Beverley and Clayton, 1993). Trypanosoma brucei rhodesiense, YTat1.1 (Ruben et al., 1983) cells

(Poon, 2012) were used for TbArl13 inducible knockout, and all endogenous tagging and inducible expression experiments in the procyclic form. TbArl13 RNAi cells were generated in procyclic Trypanosoma brucei brucei, 29.13 cells (Wirtz et al., 1999). For BSF experiments, phenotypes of TbArl13 RNAi and rescue assays were analyzed using Dlb427 (T. brucei monomorphic Lister 427 cells engineered for Tet/Cmt doubleinducible expression) (Li et al., 2017; Poon, 2012). A PCR-based endogenous tagging system (pPOT) was adopted to generate TbArl13-mNeonGreen, TbArl3A-mNeonGreen and TagRFPt-TbArl3C endogenous tagging (Dean et al., 2015).


stably engineered for tetracycline (Tet) and/or cumate (Cmt) double-inducible expression

Plasmids and antibodies Plasmids used in this study are listed in supplementary information (Table S1). Point mutations was introduced into wild type TbArl13 or TbArl3 genes by site directed mutagenesis using inverse PCR (Reikofski and Tao, 1992). For TbArl13 RNAi complementation assays, codons in full length TbArl13 coding sequence (CDS) were replaced with alternative codons whenever applicable to generate an RNAi-resistant version (iR) of TbArl13 CDS. The synthesized RNAi-resistant CDS served as PCR template from which different mutants/truncations were generated. Rabbit anti-TbArl13 polyclonal antiserum was generated using purified His-TbArl13 expressed in E. coli. Antiserum was further affinity purified on PVDF membrane strips loaded with GST-TbArl13(Ritter, 1991). Other antibodies used in this study are listed in supplementary information (Table S2).

Fluorescence and electron microscopy Unless otherwise stated, PCF and BSF cells were fixed with 4% paraformaldehyde before or after extraction with 0.25% NP-40 or Triton X-100. The fixed cells or cytoskeleton samples were blocked with 3% BSA and then incubated with primary and secondary antibodies. Images were captured either on a Zeiss Axio Observer Z1 fluorescence

FLUOVIEW FV3000 confocal microscope. For TEM, PCF and BSF cells were fixed and resin-embedded based on established protocols (Hoog et al., 2010) . Ultra-thin sections were post stained with Reynold’s lead citrate for 10 min. Transmission electron microscopy was performed using a JEM-1220 TEM from JEOL Asia, and an Orius 830 CCD camera (2K by 2K) from Gatan.


microscope with a CoolSNAP HQ2 CCD camera (Photometrics) or on an Olympus

Image analysis All images were adjusted and edited for presentation using ImageJ. For all images subject to measurement and comparison, both acquisition and image display parameters were kept constant. For flagellar fluorescence intensity quantitation, axonemes were traced using freehand tool in ImageJ (line width=3 pixels). The average intensity values along the first 2µm of the anterior axonemes were used for analysis in Fig. 2E and Fig. S5B. The anterior tip of the axonemes were determined using either the beginning of mCherry or mNeonGreen signals, whichever was stronger. Correlation coefficient was calculated using Graphpad Prism 6.

Bimolecular fluorescence complementation (BiFC) VN and VC fragments were cloned from pBiFC-VN155 (I152L) (Addgene plasmid # 27097) (Kodama and Hu, 2010a) and pCE-BiFC-VC155 (Addgene plasmid # 22020) (Shyu et al., 2008). BiFC reporter plasmids were generated by fusing TbArl13 to VN in the Tet-inducible pLEW100 vector, or TbArl3 coding sequences to VC in the Cmtinducible pDEX vector. Procyclic cells stably transfected with both VN- and VCcontaining plasmids were induced with cumate (10µg/ml) and tetracycline (20µg/ml),

Proximity-dependent biotin identification (BioID) PCF cells stably transfected with pDEX-FLiR-MycBirA were induced with 10g/ml cumate for 24 hours. These cells and WT PCF cells (negative control) were then cultured in the presence of 50 µM biotin for an additional 24 hours. The procedures for streptavidin-based pull-down of biotinylated proteins followed the published protocol (Morriswood et al., 2013). To maximize peptide recovery, captured proteins were reduced, alkylated and digested on the streptavidin-coated beads. LC-MS/MS of digested peptides were carried out on an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA) in Trap Elute configuration. Tandem MS analysis was performed on a


alone or together for 24 hours before live fluorescence imaging using YFP parameters.

TripleTOF 5600 system (AB SCIEX, Foster City, CA, USA) in Information Dependent Mode. MS spectra were acquired using the Analyst 1.6 software (AB SCIEX). Protein identification was performed with MASCOT Server 2.4.0 (Matrix Science), and data was searched against the T. brucei protein database (TbruceiTREU927, Release-28). Protein identification threshold was set at 1% FDR (false discovery rate) based on reversed protein sequences. ‘Protein content’ is calculated using the emPAI method (Ishihama et al., 2005), which estimates the weight fraction of each protein against the weight of the total pulldown mixture. Protein IDs that were found only in the TbArl13-mycBirA sample but not in the negative control sample were kept and ranked based on their relative protein content. To determine whether a protein is in the ‘flagellar proteome’, the result list was compared with past proteomics studies on T. brucei flagella (Broadhead et al., 2006; Oberholzer et al., 2011; Subota et al., 2014) using the Tritryp Strategy tool (tritrypdb.org).

Pull-downs and co-IP experiments Reciprocal in vitro pull downs of purified His-TbArl13 and GST-TbArl3A/C beads were carried out in standard protocols(Einarson et al., 2007) and were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. For pulldown of TbArl3A/B/C-VC from T. brucei lysates, His-TbArl13 coated beads were incubated with lysates of cells

15µM imidazole. As negative controls, Ni-NTA beads were incubated with untransformed BL21 bacterial lysate before incubating with the same cell lysates under same conditions. After incubation, beads were washed 3 times with PBS containing 20µM imidazole and once with PBS. Bound proteins were eluted by boiling in Laemmli buffer. Co-immunoprecipitation studies of TbArl13 with FL-YFP, DD-YFP, CD-YFP and YFP only were conducted using GFP-nAb beads (Allele Biotechnology) following manufacture’s protocol.


expressing respective transgenes for 2 hours at room temperature, in the presence of

Guanine nucleotide exchange assay and GTP binding assay Guanine nucleotide exchange assays were conducted based on established protocols (Gotthardt et al., 2015). Briefly, GST-TbArl3A and GST-TbArl3C were preloaded with mant-GDP in the presence of 50 mM EDTA and 5-fold mant-GDP (Sigma) at 20 ̊C for 90 min. The nucleotide change was stopped by adding 100 mM MgCl2. Excess nucleotides were removed by desalting columns, 7K MWCO (Thermo Scientific). 0.5 μM mant-GDP labeled GST-Arl3A or GST-Arl3C were incubated with different concentrations of HisTbArl13 and unlabeled GTP (20-fold excess) at 20 ̊C. GTP binding assay was performed using the RhoGEF exchange assay kit (Cytoskeleton, Inc) based on manufacturer’s instructions. 4 μM purified GST-Arl3A or GST-Arl3C were incubated with or without 1.5 μM His-TbArl13 in the presence of 1.5 μM mant-GTP at 20 ̊C. All fluorescence data was recorded by Tecan Infinite M200 plate reader. Both GEF and GTP binding assays were normalized with initial fluorescence reading. All experiments were repeated 6 times. Kobserved (Kobs) were obtained by fitting the data as one phase exponential decay


using Graphpad Prism 7.

Acknowledgements We wish to thank Dr. Rudolf Meier for help with bioinformatics analyses performed in this study, Drs. Samuel Dean and Jack Sunter for the pPOT constructs used in mNeonGreen and TagRFPt tagging, Dr. Brian Burke for the BioID construct, Dr. Philippe Bastin for the L3B2 antibodies, Dr. David Engman for the anti-Tb24 antibodies and Dr. Sudipto Roy for critical reading of this manuscript.

Competing interests No competing interests declared.

Funding This work was supported by Singapore Ministry of Education grants (R-154-000-A30114 and R154-000-B04-112 to CYH). AS is funded by Southeast Asian Biodiversity


Genomics grants (R-154-000-648-646 and R-154-000-648-733).

Aslett, M., Aurrecoechea, C., Berriman, M., Brestelli, J., Brunk, B. P., Carrington, M., Depledge, D. P., Fischer, S., Gajria, B., Gao, X. et al. (2010). TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Research 38, D457-D462. Badano, J. L., Mitsuma, N., Beales, P. L. and Katsanis, N. (2006). The Ciliopathies: An Emerging Class of Human Genetic Disorders. Annu Rev Genomics Hum Genet 7, 125-148. Banky, P., Huang, L. J. S. and Taylor, S. S. (1998). Dimerization/docking domain of the type I regulatory subunit of cAMP- dependent protein kinase: Requirements for dimerization and docking are distinct but overlapping. Journal of Biological Chemistry 273, 35048-35055. Bastin, P., Pullen, T. J., Moreira-Leite, F. F. and Gull, K. (2000). Inside and outside of the trypanosome flagellum:a multifunctional organelle. Microbes and infection / Institut Pasteur 2, 1865-1874. Benson, D. A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. and Sayers, E. W. (2013). GenBank. Nucleic Acids Res 41, D36-42. Beverley, S. M. and Clayton, C. E. (1993). Transfection of Leishmania and Trypanosoma brucei by electroporation. Methods Mol Biol 21, 333-48. Breslow, D. K., Koslover, E. F., Seydel, F., Spakowitz, A. J. and Nachury, M. V. (2013). An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier. The Journal of cell biology 203, 129-47. Broadhead, R., Dawe, H. R., Farr, H., Griffiths, S., Hart, S. R., Portman, N., Shaw, M. K., Ginger, M. L., Gaskell, S. J., McKean, P. G. et al. (2006). Flagellar motility is required for the viability of the bloodstream trypanosome. Nature 440, 224-227. Cantagrel, V., Silhavy, J. L., Bielas, S. L., Swistun, D., Marsh, S. E., Bertrand, J. Y., Audollent, S., Attie´-Bitach, T., Holden, K. R., Dobyns, W. B. et al. (2008). Mutations in the Cilia Gene ARL13B Lead to the Classical Form of Joubert Syndrome. American Journal of Human Genetics 83, 170-179. Carr, D. W., Fujita, A., Stentz, C. L., Liberty, G. A., Olson, G. E. and Narumiya, S. (2001). Identification of Sperm-specific Proteins that Interact with A-kinase Anchoring Proteins in a Manner Similar to the Type II Regulatory Subunit of PKA. Journal of Biological Chemistry 276, 17332-17338. Casalou, C., Seixas, C., Portelinha, A., Pintado, P., Barros, M., Ramalho, J. S., Lopes, S. S. and Barral, D. C. (2014). Arl13b and the non-muscle myosin heavy chain IIA are required for circular dorsal ruffle formation and cell migration. Journal of cell science 127, 2709-22. Caspary, T., Larkins, C. E. and Anderson, K. V. (2007). The Graded Response to Sonic Hedgehog Depends on Cilia Architecture. Developmental cell 12, 767-778. Cavalier-Smith, T. (2010). Kingdoms Protozoa and Chromista and the eozoan root of the eukaryotic tree. Biology Letters 6, 342-345. Cevik, S., Hori, Y., Kaplan, O. I., Kida, K., Toivenon, T., Foley-Fisher, C., Cottell, D., Katada, T., Kontani, K. and Blacque, O. E. (2010). Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans. Journal of Cell Biology 188, 953-969. Cevik, S., Sanders, A. A. W. M., Van Wijk, E., Boldt, K., Clarke, L., van Reeuwijk, J., Hori, Y., Horn, N., Hetterschijt, L., Wdowicz, A. et al. (2013). Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARL13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain. PLoS Genetics 9.


References


Christian, F., Szaszak, M., Friedl, S., Drewianka, S., Lorenz, D., Goncalves, A., Furkert, J., Vargas, C., Schmieder, P., Gotz, F. et al. (2011). Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes. J Biol Chem 286, 9079-96. Cuvillier, a., Redon, F., Antoine, J. C., Chardin, P., DeVos, T. and Merlin, G. (2000a). LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity. Journal of cell science 113 ( Pt 1, 2065-2074. Cuvillier, a., Redon, F., Antoine, J. C., Chardin, P., DeVos, T. and Merlin, G. (2000b). LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity. Journal of cell science 113 ( Pt 1, 2065-2074. Darriba, D., Taboada, G. L., Doallo, R. and Posada, D. (2011). ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164-5. Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E. and Gull, K. (2015). A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open biology 5, 140197-140197. Diener, D. R., Ang, L. H. and Rosenbaum, J. L. (1993). Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3. The Journal of cell biology 123, 183-190. Duldulao, N. a., Lee, S. and Sun, Z. (2009). Cilia localization is essential for in vivo functions of the Joubert syndrome protein Arl13b/Scorpion. Development (Cambridge, England) 136, 4033-4042. Einarson, M. B., Pugacheva, E. N. and Orlinick, J. R. (2007). Identification of ProteinProtein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins. CSH Protoc 2007, pdb.top11. Fansa, E. K., Kösling, S. K., Zent, E., Wittinghofer, A. and Ismail, S. (2016). PDE6δmediated sorting of INPP5E into the cilium is determined by cargo-carrier affinity. Nature communications 7, 11366. Fujita, a., Nakamura, K., Kato, T., Watanabe, N., Ishizaki, T., Kimura, K., Mizoguchi, a. and Narumiya, S. (2000). Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella. Journal of cell science 113 ( Pt 1, 103-112. Garcia-Gonzalo, F. R. and Reiter, J. F. (2012). Scoring a backstage pass: Mechanisms of ciliogenesis and ciliary access. Journal of Cell Biology 197, 697-709. Gotthardt, K., Lokaj, M., Koerner, C., Falk, N., Gießl, A. and Wittinghofer, A. (2015). A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins. eLife 4, 1-16. Grayson, C., Bartolini, F., Chapple, J. P., Willison, K. R., Bhamidipati, A., Lewis, S. A., Luthert, P. J., Hardcastle, A. J., Cowan, N. J. and Cheetham, M. E. (2002). Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3. Hum Mol Genet 11, 3065-3074. Hanke-Gogokhia, C., Wu, Z., Gerstner, C. D., Frederick, J. M., Zhang, H. and Baehr, W. (2016). Arf-like protein 3 (ARL3) regulates protein trafficking and ciliogenesis in mouse photoreceptors. Journal of Biological Chemistry 291, jbc.M115.710954. Higginbotham, H., Eom, T. Y., Mariani, L. E., Bachleda, A., Hirt, J., Gukassyan, V., Cusack, C. L., Lai, C., Caspary, T. and Anton, E. S. (2012). Arl13b in Primary Cilia Regulates the Migration and Placement of Interneurons in the Developing Cerebral Cortex. Developmental cell 23, 925-938. Hildebrandt, F., Benzing, T. and Katsanis, N. (2011). Ciliopathies. New England Journal of Medicine 364, 1533-1543.


Hoog, J. L., Gluenz, E., Vaughan, S. and Gull, K. (2010). Ultrastructural investigation methods for Trypanosoma brucei. Methods Cell Biol 96, 175-96. Hori, Y., Kobayashi, T., Kikko, Y., Kontani, K. and Katada, T. (2008). Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation. Biochemical and Biophysical Research Communications 373, 119-124. Hu, Q., Milenkovic, L., Jin, H., Scott, M. P., Nachury, M. V., Spiliotis, E. T. and Nelson, W. J. (2010). A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution. Science 329, 436-439. Humbert, M. C., Weihbrecht, K., Searby, C. C., Li, Y., Pope, R. M., Sheffield, V. C. and Seo, S. (2012). ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting. Proceedings of the National Academy of Sciences of the United States of America 109, 19691-6. Hundsrucker, C. and Klussmann, E. (2008). Direct AKAP-mediated protein-protein interactions as potential drug targets. Handb Exp Pharmacol, 483-503. Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J. and Mann, M. (2005). Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4, 1265-72. Ishikawa, H. and Marshall, W. F. (2011). Ciliogenesis: building the cell's antenna. Nature Reviews Molecular Cell Biology 12, 222-234. Ismail, S. a., Chen, Y.-X., Rusinova, A., Chandra, A., Bierbaum, M., Gremer, L., Triola, G., Waldmann, H., Bastiaens, P. I. H. and Wittinghofer, A. (2011). Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo. Nature chemical biology 7, 942-949. Ivanova, A. A., Caspary, T., Seyfried, N. T., Duong, D. M., West, A. B., Liu, Z. and Kahn, R. A. (2017). Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF). J Biol Chem 292, 11091-11108. Kahn, R. A., East, M. P. and Francis, J. W. (2014). ARF-Like (ARL) Proteins. In Ras Superfamily Small G Proteins: Biology and Mechanisms 2: Transport, (ed. A. Wittinghofer), pp. 215-251. Cham: Springer International Publishing. Katoh, K. and Standley, D. M. (2013). MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol Biol Evol 30, 772-80. Kodama, Y. and Hu, C. D. (2010a). An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Biotechniques 49, 793-805. Kodama, Y. and Hu, C. D. (2010b). An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Biotechniques 49, 793-803. Kohl, L., Robinson, D. and Bastin, P. (2003). Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes. EMBO Journal 22, 5336-5346. Kohl, L., Sherwin, T. and Gull, K. (1999). Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle. The Journal of Eukaryotic Microbiology 46, 105-109. Kosling, S. K., Fansa, E. K., Maffini, S. and Wittinghofer, A. (2018). Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment. Biol Chem. Kück, P., Meusemann, K., Dambach, J., Thormann, B., von Reumont, B. M., Wägele, J. W. and Misof, B. (2010). Parametric and non-parametric masking of randomness in sequence alignments can be improved and leads to better resolved trees. Frontiers in Zoology 7, 10.


Larkins, C. E., Aviles, G. D. G., East, M. P., Kahn, R. A. and Caspary, T. (2011). Arl13b regulates ciliogenesis and the dynamic localization of Shh signaling proteins. Molecular biology of the cell 22, 4694-703. Lester, L. B. and Scott, J. D. (1997). Anchoring and scaffold proteins for kinases and phosphatases. Recent Prog Horm Res 52, 409-29; discussion 429-30. Li, F.-J., Xu, Z.-S., Aye, H. M., Brasseur, A., Lun, Z.-R., Tan, K. S. W. and He, C. Y. (2017). An efficient cumate-inducible system for procyclic and bloodstream form Trypanosoma brucei. Molecular and biochemical parasitology 214, 101-104. Li, Y., Wei, Q., Zhang, Y., Ling, K. and Hu, J. (2010). The small GTPases ARL-13 and ARL-3 coordinate intraflagellar transport and ciliogenesis. Journal of Cell Biology 189, 1039-1051. Lin, Y.-C., Niewiadomski, P., Lin, B., Nakamura, H., Phua, S. C., Jiao, J., Levchenko, A., Inoue, T., Rohatgi, R. and Inoue, T. (2013). Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nature chemical biology 9, 437-43. Lokaj, M., Kösling, S. K., Koerner, C., Lange, S. M., Van Beersum, S. E. C., Van Reeuwijk, J., Roepman, R., Horn, N., Ueffing, M., Boldt, K. et al. (2015). The Interaction of CCDC104/BARTL1 with Arl3 and Implications for Ciliary Function. Structure 23, 2122-2132. Lu, H., Tiak, M., Narasimhan, V., Kumar, S., Choksi, S. P. and Roy, S. (2015). A function for the Joubert syndrome protein Arl13b in ciliary membrane extension and ciliary length regulation. Developmental biology 397, 225-236. Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1162-1164. Mariani, L. E., Bijlsma, M. F., Ivanova, A. I., Suciu, S. K., Kahn, R. A. and Caspary, T. (2016). Arl13b regulates Shh signaling from both inside and outside the cilium. Molecular biology of the cell. Miertzschke, M., Koerner, C., Spoerner, M. and Wittinghofer, A. (2014). Structural insights into the small G-protein Arl13B and implications for Joubert syndrome. Biochem. J 457, 301-311. Misof, B. and Misof, K. (2009). A Monte Carlo Approach Successfully Identifies Randomness in Multiple Sequence Alignments : A More Objective Means of Data Exclusion. Syst Biol 58, 21-34. Morriswood, B., Havlicek, K., Demmel, L., Yavuz, S., Sealey-Cardona, M., Vidilaseris, K., Anrather, D., Kostan, J., Djinović-Carugo, K., Roux, K. J. et al. (2013). Novel bilobe components in Trypanosoma brucei identified using proximity-dependent biotinylation. Eukaryotic cell 12, 356-367. Nachury, M. V. (2011). Trafficking to the Ciliary Membrane. 59-87. Nozaki, S., Katoh, Y., Terada, M., Michisaka, S., Funabashi, T., Takahashi, S., Kontani, K. and Nakayama, K. (2017). Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E. J Cell Sci 130, 563-576. Oberholzer, M., Langousis, G., Nguyen, H. T., Saada, E. a., Shimogawa, M. M., Jonsson, Z. O., Nguyen, S. M., Wohlschlegel, J. a. and Hill, K. L. (2011). Independent analysis of the flagellum surface and matrix proteomes provides insight into flagellum signaling in mammalianinfectious Trypanosoma brucei. Molecular & cellular proteomics : MCP 10, M111.010538. Poon, S. K. (2012). A modular and optimized single marker system for generating. 2. Povelones, M. L., Tiengwe, C., Gluenz, E., Gull, K., Englund, P. T. and Jensen, R. E. (2013). Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1. Mol Microbiol 87, 713-29.


Price, H. P., Panethymitaki, C., Goulding, D. and Smith, D. F. (2005). Functional analysis of TbARL1, an N-myristoylated Golgi protein essential for viability in bloodstream trypanosomes. Journal of cell science 118, 831-841. Ralston, K. S. and Hill, K. L. (2006). Trypanin, a component of the flagellar dynein regulatory complex, is essential in bloodstream form African trypanosomes. PLoS pathogens 2, 0873-0882. Ralston, K. S., Lerner, A. G., Diener, D. R. and Hill, K. L. (2006). Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system. Eukaryot Cell 5, 696-711. Rambaut, A., Suchard, M. A., Xie, D. and Drummond, A. J. (2015). Tracer v1. 6. 2014. Reikofski, J. and Tao, B. Y. (1992). Polymerase chain reaction (PCR) techniques for sitedirected mutagenesis. Biotechnol Adv 10, 535-47. Reiter, J. F., Blacque, O. E. and Leroux, M. R. (2012). The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO reports 13, 608-618. Ritter, K. (1991). Affinity purification of antibodies from sera using polyvinylidenedifluoride (PVDF) membranes as coupling matrices for antigens presented by autoantibodies to triosephosphate isomerase. J Immunol Methods 137, 209-15. Robinson, D. R., Sherwin, T., Ploubidou, A., Byard, E. H. and Gull, K. (1995). Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. Journal of Cell Biology 128, 1163-1172. Ronquist, F. and Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-4. Roy, K., Jerman, S., Jozsef, L., McNamara, T., Onyekaba, G., Sun, Z. and Marin, E. P. (2017). Palmitoylation of the ciliary GTPase ARL13b is necessary for its stability and its role in cilia formation. J Biol Chem 292, 17703-17717. Ruben, L., Egwuagu, C. and Patton, C. L. (1983). African trypanosomes contain calmodulin which is distinct from host calmodulin. Biochim Biophys Acta 758, 104-13. Rubin, C. S. (1994). A kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim Biophys Acta 1224, 467-79. Sahin, A., Lemercier, G., Tetaud, E., Espiau, B., Myler, P., Stuart, K., Bakalara, N. and Merlin, G. (2004). Trypanosomatid flagellum biogenesis: ARL-3A is involved in several species. Experimental Parasitology 108, 126-133. Schrick, J. J., Vogel, P., Abuin, A., Hampton, B. and Rice, D. S. (2006). ADP-ribosylation factor-like 3 is involved in kidney and photoreceptor development. The American journal of pathology 168, 1288-98. Scott, J. D., Mangili, J. A., Stofkos, E., Randy, J., Comer, D., Vitalisll, a., I, M. E. D. S. C. I. and Albert, R. (1990). Type II Regulatory Subunit Dimerization Determines Localization of the CAMP-dependent Protein Kinase * the Subcellular. Journal of Biological Chemistry 265, 2156121566. Seixas, C., Choi, S. Y., Polgar, N., Umberger, N. L., East, M. P., Zuo, X., Moreiras, H., Ghossoub, R., Benmerah, A., Kahn, R. A. et al. (2015). Arl13b and the exocyst interact synergistically in ciliogenesis. Molecular biology of the cell, mbc.E15-02-0061-. Shyu, Y. J., Hiatt, S. M., Duren, H. M., Ellis, R. E., Kerppola, T. K. and Hu, C. D. (2008). Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis. Nat Protoc 3, 588-96.


Sivadas, P., Dienes, J. M., Maurice, M. S., Meek, W. D. and Yang, P. (2012). A flagellar a-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex. Journal of Cell Biology 199, 639-651. Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies. Bioinformatics 30, 1312-3. Stothard, P. (2000). The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28, 1102, 1104. Subota, I., Julkowska, D., Vincensini, L., Reeg, N., Buisson, J., Blisnick, T., Huet, D., Perrot, S., Santi-Rocca, J., Duchateau, M. et al. (2014). Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sublocalisation and dynamics. Molecular & cellular proteomics : MCP, 1769-1786. Sun, Z., Amsterdam, A., Pazour, G. J., Cole, D. G., Miller, M. S. and Hopkins, N. (2004). A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development (Cambridge, England) 131, 4085-93. Sung, C.-H. and Leroux, M. R. (2013). The roles of evolutionarily conserved functional modules in cilia-related trafficking. Nature cell biology 15, 1387-97. Tröger, J., Moutty, M. C., Skroblin, P. and Klussmann, E. (2012). A-kinase anchoring proteins as potential drug targets. Br J Pharmacol 166, 420-33. Tyler, K. M., Fridberg, A., Toriello, K. M., Olson, C. L., Cieslak, J. a., Hazlett, T. L. and Engman, D. M. (2009). Flagellar membrane localization via association with lipid rafts. Journal of cell science 122, 859-866. Vaughan, S., Kohl, L., Ngai, I., Wheeler, R. J. and Gull, K. (2008). A Repetitive Protein Essential for the Flagellum Attachment Zone Filament Structure and Function in Trypanosoma brucei. Protist 159, 127-136. Wallace, I. M., O'Sullivan, O., Higgins, D. G. and Notredame, C. (2006). M-Coffee: combining multiple sequence alignment methods with T-Coffee. Nucleic Acids Research 34, 1692-1699. Wang, Y., Ho, T. G., Bertinetti, D., Neddermann, M., Franz, E., Mo, G. C. H., Schendowich, L. P., Sukhu, A., Spelts, R. C., Zhang, J. et al. (2014). Isoform-selective disruption of AKAP-localized PKA using hydrocarbon stapled peptides. ACS Chemical Biology 9, 635-642. Wheeler, R. J., Scheumann, N., Wickstead, B., Gull, K. and Vaughan, S. (2013). Cytokinesis in trypanosoma brucei differs between bloodstream and tsetse trypomastigote forms: Implications for microtubule-based morphogenesis and mutant analysis. Molecular microbiology 90, 1339-1355. Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. (1999). A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99, 89-101. Xu, X., Olson, C. L., Engman, D. M. and Ames, J. B. (2012). NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei. Protein Science 21, 19421947. Xue, Y., Chen, H., Jin, C., Sun, Z. and Yao, X. (2006). NBA-Palm: prediction of palmitoylation site implemented in Naïve Bayes algorithm. BMC Bioinformatics 7, 458. Zhang, Q., Li, Y., Zhang, Y., Torres, V. E., Harris, P. C., Ling, K. and Hu, J. (2016). GTPbinding of ARL-3 is activated by ARL-13 as a GEF and stabilized by UNC-119. Scientific Reports 6, 24534. Zhou, C., Cunningham, L., Marcus, A. I., Li, Y. and Kahn, R. A. (2006). Arl2 and Arl3 regulate different microtubule-dependent processes. Molecular biology of the cell 17, 24762487.


Zhou, Q., Liu, B., Sun, Y. and He, C. Y. (2011). A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei. Journal of cell science 124, 3848-3858.

Fig. 1 TbArl13 associates with the flagellar axoneme. (A) Domain organization of TbArl13 and human Arl13b. (B) Endogenously expressed TbArl13-YFP-3HA (labeled by anti-HA; left) and native TbArl13 (labeled by anti-TbArl13; right) are both present in 0.25% NP40-extracted flagella. The cells are counter-stained with DAPI (blue) to visualize the DNA-containing nucleus (large oval) and kinetoplast (small oval near the base of each flagellum). Cells in different cell cycle stages, with single or duplicated organelles are shown. (C) The polyclonal anti-TbArl13 antibodies specifically recognize


Figures

native TbArl13 and TbArl13-YFP-3HA on immunoblots. (D) Cells stably expressing YFP-RSP3 (radial spoke protein 3, Tb927.11.1150; red) in the axoneme were extracted with 0.25%NP40, fixed and labeled with anti-TbArl13 (green), YL1/2 for the basal bodies (BB; magenta) and DAPI (blue). Enlarged views of the proximal region of one flagellum (demarcated by dashed line) are shown on the right. Note that both TbArl13 (green arrow) and YFP-RSP3 (red arrow) initiate at the same site. (E) Representative plot profiles of fluorescence intensities of both YFP-RSP3 (red) and anti-TbArl13 (green) along the axoneme, demonstrating gradient distribution of TbArl13 along the axoneme (F) NP40-extracted cytoskeleton were co-stained with anti-PAR antibody to mark the paraflagellar rod (PFR; red), YL1/2 (BB, magenta) and TbArl13 (green). Enlarged views of the proximal ends of a new flagellum (box 1) and a nascent flagellum formed just posterior to an existing flagellum (box 2) are shown on the right. The anti-TbArl13 signals initiate at a position (green arrow) significantly closer to the BB than that of PFR (red arrow). Anti-TbArl13 also stains the nascent flagellum (arrowhead), where no PFR is yet assembled. (G) Schematic drawing of the trypanosome flagellum, highlighting the


relative position of the PFR and axoneme to the basal body. All scale bars = 5μm.


Fig. 2 TbArl13 is targeted to the flagellum via its D/D domain. (A) Cells with inducible expression of YFP-tagged full length (FL-YFP) or truncated TbArl13 (DDYFP, CD-YFP) were fixed with 4% PFA, with or without prior extraction with 0.25% TritonX-100. Insets focus on the proximal end of the flagellum of a representative cell, highlighting the enrichment of both FL-YFP and DD-YFP. (B) Co-immunoprecipitation of endogenous TbArl13 with FL-YFP, DD-YFP but not with CD-YFP or YFP alone. GFP-nAb beads were incubated with lysates from cells expressing FL-YFP, DD-YFP, CD-YFP or YFP alone. Anti-TbArl13 was used to detect the presence of native TbArl13 in Input (10%), co-immunoprecipitated (IP) and unbound (UB) fractions. (C-D) Fluorescence images of 0.25% NP40 extracted (C) or live cells (D) with endogenous expression of TbArl13-mNeonGreen (green) and varying levels of mCherry-DD (red). (E) Quantitation of mCherry-DD and TbArl13-mNeonGreen intensities in 21 extracted flagella as those shown in C. (F) Cell fractionation assay using 1% NP40 shows reduced level of TbArl13-mNeonGreen in the cytoskeletal fraction (P, pellet) when mCherry-DD expression is induced (Cmt+). The soluble fraction (S, supernatant) did not show


observable changes. All scale bars = 5μm.

iKO cells at 0 hr (control), 48hrs and 72hrs upon tetracycline removal were labeled with anti-PFR1 for PFR (red), L3B2 for FAZ (Green), and DAPI (blue). Cells with shortened FAZ (arrowheads) and detached/shortened flagella (arrows) are observed. Scale bars =


Fig. 3 TbArl13 is essential for flagellum biogenesis and cell survival. (A) TbArl13

5μm (B, C) Statistical analyses of PFR and FAZ length measurements. Single flagellated cells were measured for each time point (n=99, 92, 100, 95 for 0hr, 48hrs, 72hrs and WT respectively), and the results are shown as mean value (thick bar) ± SD (thin error bars). Two tailed student t-test was used to evaluate the significance of difference between the test groups and the control group (iKO 0hr). ****: p

The unusual flagellar targeting mechanism and functions of the

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