Submitted to Open Biology: FOR REVIEW ONLY A centriolar FOP-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes Journal: Open Biology Manuscript ID RSOB-17-0218.R2 Article Type: Research Date Submitted by the Author: 31-May-2018 Complete List of Authors: Harmer, Jane; Lancaster University, Divison of Biomedical and Life Sciences; University of Huddersfield School of Applied Sciences, Department of Biological Sciences Towers, Katie; OXford Brookes University, Department of Biological and Medical Sciences Addison, Max; Lancaster University, Divison of Biomedical and Life Sciences Vaughan, Sue; OXford Brookes University, Department of Biological and Medical Sciences Ginger, Michael; University of Huddersfield School of Applied Sciences, Department of Biological Sciences McKean, Paul; Lancaster University, Divison of Biomedical and Life Sciences Subject: cellular biology, microbiology basal body, ciliogenesis, FOP, Trypanosoma brucei, cell morphogenesis, Keywords: paraflagellar rod http://mc.manuscriptcentral.com/rsob Page 1 of 32 Submitted to Open Biology: FOR REVIEW ONLY 1 A centriolar FOP-like protein required for paraflagellar rod assembly, but 2 not axoneme assembly in African trypanosomes 3 4 Jane Harmer1,4, Katie Towers2, Max Addison1, Sue Vaughan2, Michael L. Ginger3*, Paul G. 5 McKean1* 6 7 1Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, 8 Lancaster, LA1 4YQ, UK 9 2Department of Biological and Medical Sciences, Faculty of Health and Life Science, Oxford Brookes 10 University, Gipsy Lane, Oxford, OX3 0BP, UK 11 3Department of Biological and Geographical Sciences, School of Applied Sciences, University of 12 Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK 13 4Present address: Department of Biological and Geographical Sciences, School of Applied Sciences, 14 University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK 15 16 Authors for correspondence: 17 Michael L. Ginger email: [email protected] 18 Paul G. McKean email: [email protected] 19 *MLG and PGM are co-senior authors 20 Running head: Trypanosome FOPL function 21 Subject Area: cellular biology/microbiology 22 Keywords: basal body; ciliogenesis; FOP; Trypanosoma brucei, cell morphogenesis, paraflagellar 23 rod 1 http://mc.manuscriptcentral.com/rsob Submitted to Open Biology: FOR REVIEW ONLY Page 2 of 32 24 25 Abstract 26 Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organising centres 27 (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the 28 axoneme extends. We report conservation of FOP family proteins, TbFOPL and TbOFD1, in the 29 evolutionarily divergent sleeping sickness parasite Trypanosoma brucei, showing in contrast to 30 mammalian cells where FOP is essential for flagellum assembly, depletion of a trypanosome FOP 31 homologue, TbFOPL, affects neither axoneme nor flagellum elongation. Instead, TbFOPL depletion 32 causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the 33 paraflagellar rod (PFR). That depletion of centriolar TbFOPL causes failure in PFR assembly is 34 surprising since PFR nucleation commences ~2 µm distal from the basal body. When over-expressed 35 with a C-terminal myc-epitope, TbFOPL was also observed at mitotic spindle poles. Little is known 36 about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible 37 additional MTOC function for TbFOPL parallels MTOC localisation of FOPlike protein 38 TONNEAU1 in acentriolar plants. More generally, our functional analysis of TbFOPL emphasizes 39 significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss 40 how at the molecular level FOP homologues may contribute to flagellum assembly and function in 41 diverse flagellates. 42 2 http://mc.manuscriptcentral.com/rsob Page 3 of 32 Submitted to Open Biology: FOR REVIEW ONLY 43 1. Introduction 44 Coupled, N-terminally located TOFLisH motifs define a small family of eukaryotic proteins, the FOP 45 family; members of which are required for ciliogenesis in flagellate eukaryotes, and cortical 46 cytoskeleton organisation in plant cells. Family members conserved amongst flagellate eukaryotes are 47 FOP (standing for FGFR1 oncogene partner), OFD1 (mutated in orofaciodigital syndrome 1), and 48 FOR20 (or FOP-related protein of 20kDa) (1-8). With regard to localisation, in animal cells, OFD1, 49 FOR20, and FOP are all found at the base of cilia associated with basal bodies (or centrioles), either at 50 the level of the triplet microtubule barrel or the transition zone. The microtubule axoneme (the 51 defining structure of all eukaryotic flagella or cilia) extends from the basal body; the transition zone 52 defines the most proximal region of the flagellum and exhibits its own particular architecture, where 53 Y-shaped projections link axoneme outer-doublet microtubules to the flagellar membrane (9). FOP, 54 OFD1 and FOR20 are all required for the formation of a primary cilium; assembled by many types of 55 animal cell in response to appropriate environmental cues (10). Roles for FOR20 and OFD1 in cilia 56 assembly have been described in the ciliates Paramecium tetraurelia and Tetrahymena thermophila 57 (5, 11, 12), but we are not aware of any reports regarding functional studies of candidate OFD1 and 58 FOP orthologues in other flagellate protists or fungi. 59 One member of the FOP protein family is also conserved in at least one group of aflagellate 60 eukaryotes, land-plants. The protein TONNEAU1 (or TON1), which is most similar to FOP, interacts 61 with at least one classic protein found at microtubule organising centres, centrin, and is required for 62 organisation of cortical microtubules during cell elongation and division (13). Absence of 63 TONNEAU1 from Arabidopsis, an aflagellate angiosperm, or the evolutionarily more basal bryophyte 64 moss Physcomitrella patens, which deploys flagellate motile sperm for reproduction, results in 65 organelle mis-positioning and defective development (13, 14). Another group of organisms in which a 66 cortical-based microtubule cytoskeleton exerts an overarching and relatively well understood effect on 67 cell morphogenesis and division are the flagellate trypanosomatids (15-17). Long known as the 68 aetiological agents of a variety of serious tropical diseases (e.g. African sleeping sickness, Chagas 69 disease, leishmaniasis), the parasitic trypanosomatid family belong to the excavate group of protists; 3 http://mc.manuscriptcentral.com/rsob Submitted to Open Biology: FOR REVIEW ONLY Page 4 of 32 70 which is widely recognised as evolutionarily divergent in comparison with other eukaryotic groups 71 (18, 19). 72 Flagellum assembly and function has been widely studied in trypanosomatids; many facets of this 73 biology are conserved with other flagellate eukaryotes, but there are also notable differences. Visually 74 the most notable difference is the presence of a complex paraflagellar rod (PFR). Within the flagellar 75 compartment, trypanosomatids and their nearest relatives build an elaborate extra-axonemal PFR 76 structure, consisting of a trilaminar lattice composed of proximal, intermediate and distal domains 77 (20). The PFR is attached to outer-doublet microtubules 4-7 of a canonical ‘9+2’ microtubule 78 axoneme and runs alongside the axoneme for much of the length of the flagellum. The PFR is 79 essential for flagellar beating and thus cell motility (21), and in Trypanosoma species PFR assembly 80 is required for attachment of the flagellum to the cell body, which in turn is required for normal cell 81 morphogenesis (22-23). In T. brucei, the PFR is present and attached to the axoneme from the point 82 where the flagellum exits the cell body (~2µm distant from the basal body), however, structural and/or 83 molecular cues that define assembly and asymmetric PFR attachment are not understood. 84 At a molecular level, in addition to obvious proteomic differences relating to PFR assembly, there are 85 examples of conserved proteins which have an essential flagellum assembly role that are somewhat 86 different in trypanosomatids; for instance, the tubulin cofactor C (TBCC) domain-containing protein 87 RP2 (24, 25). Here, the catalytic TBCC domain of the trypanosome RP2 protein uniquely lies 88 downstream of twinned TOFLisH motifs essential for centriolar targeting of FOP family proteins 89 (24); whereas human RP2 is targeted to the basal body by a post translation N-acyl modification (26). 90 As part of our ongoing studies looking at the role of TbRP2 in T. brucei flagellum assembly, we 91 turned our attention to the function of other trypanosomatid proteins that encode N-terminal TOF- 92 LisH motifs. We report here the conservation of OFD1 and FOP-like proteins in trypanosomatid 93 protists, and our unexpected observation that a T. brucei FOP-like protein is essential for assembly of 94 the extra-axonemal PFR, but not the axoneme itself. Our data illustrates unexpected functional and 95 evolutionary diversity in the role of conserved centriole-targeted proteins in eukaryotic flagellum 96 assembly and function. 4 http://mc.manuscriptcentral.com/rsob Page 5 of 32 Submitted to Open Biology: FOR REVIEW ONLY 97 98 2. Materials and Methods 99 2.1 Cell culture and transfection 100 Procyclic T. brucei (927Smox (27) and S427) were cultured in SDM-79 medium supplemented with 101 10% v/v foetal bovine serum and haemin