Trypanosome morphogenesis involves recruitment of a 2 pleckstrin homology domain protein by an orphan kinesin 3 to the microtubule quartet 4 5

Kinesins are motor proteins found in all eukaryotic lineages that move along microtubule tracks to mediate numerous cellular processes such as mitosis and intracellular transport of cargo. In trypanosomatids, the kinesin protein superfamily has undergone a prominent expansion, giving these protists one of the most diverse kinesin repertoires. This has led to the emergence of two trypanosomatid-restricted groups of kinesins. Here, we characterize in Trypanosoma brucei TbKifX2, a hitherto orphaned kinesin that belongs to one of these groups. Representing a rare instance, TbKifX2 tightly interacts with TbPH1, a kinesin-like protein with an inactive motor domain. TbPH1 is named after a pleckstrin homology (PH) domain present within its carboxy-terminal tail. TbKifX2 recruits TbPH1 to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that is part of the multipartite flagellum attachment zone (FAZ) and extends from the basal body to the anterior of the cell body. The proximal proteome of TbPH1 is comprised of four proteins that localize to the FAZ, consistent with the notion that the TbKifX2/TbPH1 complex are the first identified proteins to bind the MtQ along its whole length. Simultaneous ablation of both TbKifX2 and TbPH1 leads to the formation of prominent protrusions from the cell posterior. Thus, these two trypanosomatid-restricted proteins, which specifically localize to the MtQ in a microtubule-rich cell, appear to be contributors to morphogenesis in T. brucei.Trypanosomatids are a group of unicellular parasites that infect a wide range of hosts from land plants to animals. They are also eukaryotes that have been shaped by prolonged independent evolution since this domain of life has radiated from a common ancestor almost 2 billion years ago. Thus, any resulting unique biological properties can be potentially exploited for treatment of infectious diseases caused by trypanosomatids. The cytoskeleton of trypanosomatids represents an ancient organelle that has undergone such modification. Here, we show that two trypanosomatid-specific proteins named TbPH1 and TbKifX2 form a complex that localizes to the microtubule quartet, a cytoskeletal structure characteristic to trypanosomatids. Ablation of these proteins in Trypansoma brucei leads to distinct morphological defects, making them not only intrinsically interesting topics of study, but potential therapeutic targets as well.

trypanosomatids, the kinesin protein superfamily has undergone a prominent expansion, giving 21 these protists one of the most diverse kinesin repertoires. This has led to the emergence of two 22 trypanosomatid-restricted groups of kinesins. Here, we characterize in Trypanosoma brucei TbKifX2, 23 a hitherto orphaned kinesin that belongs to one of these groups. TbKifX2 tightly interacts with 24 TbPH1, a kinesin-like protein named after a pleckstrin homology (PH) domain present within its

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The only place where the continuity of the T. brucei MT array is interrupted is at the flagellar pocket, 58 the deep invagination where the flagellum exits the cell body and the only place where endo-and was processed in parallel. A total of 1,131 proteins were detected with high confidence (Andromeda 219 Protein Score ≥20; >1 unique peptide per protein) (S2 dataset). Following imputation of missing 220 values, seven proteins were shown to be preferentially biotinylated by TbPH1-BioID2-HA, as 221 visualised by plotting the -10 log t-test p value versus the t test difference in a volcano plot (Fig. 4D).
222 Statistically significant hits are found above the cut-off curve in this plot.

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TbKifX2 was the most enriched protein based on both parameters, consistent with its strong 224 interaction with TbPH1. As expected, TbPH1 is also among the enriched proteins, indicating that 225 TbKifX-2 expressing cells to visualize any potential localization to discrete cytoskeletal structures.

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Indeed both proteins co-localized to a long structure running along the long-axis of the cytoskeleton 257 adjacent to the flagellum (Fig. 5A). Furthermore, detergent extraction solubilized the cell body signal 258 ( Fig. 3D), confirming it represented the cytosolic population of TbPH1 and TbKifX2 (Figs 2A and 3C).

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We proceeded to identify specifically which cytoskeletal element TbPH1 and TbKifX2 associate with.

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We used antibody recognizing TbKifX2-HA as a proxy for the TbKifX2-TbPH1 complex because of 261 their demonstrated tight interaction (Fig 3), and their co-localization on detergent-extracted 262 cytoskeletons (Fig. 5A). We found TbKifX2-HA to be juxtaposed with the FAZ filament, although the 263 former extended further toward the proximal region of the flagellum (Fig. 5B)  and TbPH1-V5 in both cytoskeletal and cytosolic fractions in cells grown in the absence of 294 tetracycline, in which neither protein was downregulated (Fig. 6B). However, TbPH1 was 295 preferentially depleted from the cytoskeleton fraction upon TbKifX2 silencing. This result indicates 296 that TbKifX2 recruits TbPH1-V5 to the MtQ (Fig. 6B).

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Since depletion of either TbKifX2 or TbPH1 alone did not yield any other discernible phenotype, we 298 decided to deplete both proteins simultaneously. The cell line, in which both proteins were 299 downregulated, showed more pronounced growth inhibition than the single knock-downs (Fig. 6A). for other phenotypes that would explain the decreased fitness of these cells.
14 317 We assayed the MtQ and FAZ after 5 days of RNAi depletion and observed no defects in either of 318 these structures, indicating that TbPH1 and TbKifX2 do not play a role in their biogenesis (Fig. 6C).

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The lack of an effect on these structures is also consistent with the notion that these proteins do not 320 have a role in cytokinesis. However, by phase contrast microscopy we noticed the emergence of two 321 morphological phenotypes, namely multiple cells joined together on their posterior end, a 322 phenotype resembling abscission defects [51,52] and single cells with protrusions from the posterior 323 end (Fig. 6D).

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To better visualize these phenotypes, we observed these cells by scanning electron microscopy ( Fig.   325 7). While control cells showed normal trypomastigote morphology and cytokinesis ( Fig. 7), we 326 observed concatenated cells as well as cells with multiple protrusions (arrows) originating from the 327 posterior end (Fig. 7). It should be noted that this stunning phenotype, while frequently observed, 328 did not emerge in all RNAi-induced cells. The occurrence of these cell types was also corroboratively 329 found in transmission electron microscopy, which in addition provided evidence that these 330 protrusions appear to contain normal cellular contents and were surrounded by subpellicular MTs 331 (S5A Fig.). Moreover, the flagellar pocket region appeared to be unaffected by depletion of 332 TbPH1/TbKifX2 (S5B and C Fig.).

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In summary, we propose the following model (Fig. 8) for approximately 30 minutes before being transferred to microscopy slides (ThermoScientific).

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Following neutralisation in 0.1M glycine in PBS, slides were incubated in methanol at -20° C 503 overnight for permeabilisation. Slides were then rehydrated in PBS and blocked in 1% BSA in PBS for 504 1 hour followed by incubation with primary antibody for 1 hour in a humid chamber. The slides were 505 then washed three times with PBS before incubation in AlexaFluor-conjugated secondary antibody 506 (goat anti-rabbit/mouse, used at 1:1,000; Invitrogen). Following three further washes in PBS, a drop 507 of ProLong Gold Antifade reagent with DAPI (ThermoScientific) was added, a coverslip applied and 508 the slide sealed with nail polish. Slides were imaged with a Zeiss Axioscope or an Olympus FluoView 509 Fv1000 confocal microscope.

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For immunofluorescence analysis of cells extracted using the 'MT sieving' fractionation method 511 described above the pellet fraction P2 was fixed in 2.3% PFA as described. All subsequent steps were 512 the same as for whole cell immunofluorescence.

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To extract cytoskeletons conventionally, approximately 1x 10 7 PCF cells were collected and 514 centrifuged at room temperature for 5 min at 800 x g and the supernatant discarded. Thereafter, the 515 cell pellet was washed once in PBS and after resuspension in PBS applied in a drop-wise fashion to 516 Superfrost plus® slides (ThermoScientific). The cells were left to settle before the addition of PEME 517 buffer (100 mM Pipes, pH 6.9, 1 mM MgSO 4 , 2 mM EGTA, 0.1 mM EDTA) containing 0.5% (v/v) NP-40 518 (Igepal) for 10 seconds. This cytoskeleton extraction was followed by a 10 minute fixation step in 4% 519 PFA/-20°C methanol. All subsequent steps were as described above for whole cell 520 immunofluorescence.

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Another centrifugation step at 16,000 x g for 10 minutes created supernatant S2. Both supernatants, 558 S1 and S2, were then incubated with streptavidin-conjugated Dynabeads (company) for 4 hours at 4 559°C. An aliquot of flow through samples F1 and F2 were retained for western blotting and the 560 dynabeads were washed five times with PBS. A small sample of the beads was then resuspended in 561 2x SDS PAGE buffer and boiled for initial western blot analysis. For mass spectrometry analysis of the 562 TbPH1 proxisome, the procedure was repeated as described above, but only the S1 fraction was 563 further processed.