Generation of Tb<i>GAPDHL</i> and Tb<i>PGKL</i> procyclic null mutants.

<p>(<b>A</b>) Generation of Tb<i>GAPDHL</i> null mutants. (<b>B</b>) Generation of Tb<i>PGKL</i> null mutants. Cartoon schematics denote gene loci annotated with HindIII (H) restriction sites for (i) wild-type loci; (ii) loci following gene disruption and (iii) following endogenous gene-tagging with GFP (Tb<i>GAPDHL</i> only). Southern analysis of genomic DNA digested overnight at 37°C with HindIII shows blots probed sequentially with either coding sequence from the targeted gene (probe o) or sequence from the 3′ intergenic region (probe u). Relative positions of the probes are shown in the cartoon schematics. In (<b>A</b>) the order of lanes is 1, wild-type <i>GAPDHL</i>^+/+<i>T. brucei</i>; 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4-5, <i>GAPDHL</i>^+/− heterozygotes from lanes 2 and 3, respectively, in which endogenous tagging of the remaining wild-type allele results in expression of a recombinant e<i>GFP:GAPDHL</i>; 6, a <i>GAPDHL^−/−</i> mutant obtained from the stable transformation of the phleomycin-resistant heterozygote cells from lane 2. In (<b>B</b>) the order of lanes is 1, wild-type <i>PGKL</i>^+/+, 2, heterozygous <i>GAPDHL</i>^+/− cells resistant to phleomycin; 3, heterozygous <i>GAPDHL</i>^+/− cells resistant to blasticidin/HCl; 4–5, independently obtained <i>PGKL</i>^−/− mutants derived from the stable transformation of heterozygous cell lines analyzed in lanes 2 and 3, respectively.</p

PFR localization of <i>Tb</i>GAPDHL.

<p>(<b>A</b>) Localization of GFP::<i>Tb</i>GAPDHL in procyclic <i>T. brucei</i> cells. (<b>B</b>) Indirect immunofluorescence of detergent-extracted cytoskeletons using the monoclonal antibody L8C4 to detect the major PFR protein PFR2 suggests GFP::<i>Tb</i>GAPDHL is a novel PFR component. Insets 1 and 2 indicate that at the proximal end of the flagellum PFR2 incorporation into flagellar skeleton does not begin prior to GFP::<i>Tb</i>GAPDHL incorporation – <i>cf</i> inset 2 in (<b>C</b>). (<b>C</b>) A lack of co-localization in indirect immunofluorescence of cytoskeletons using the monoclonal antibody L3B2 indicates GFP::<i>Tb</i>GAPDHL is not a cytoplasmic FAZ component: inset 1 indicates flagellar GFP::<i>Tb</i>GAPDHL fluorescence extends beyond the end of the cell body as denoted by L3B2 labelling of the cytoplasmic FAZ filament; inset 2 highlights how assembly the cytoplasmic FAZ filament detected by L3B2 initiates before assembly of GFP::<i>Tb</i>GAPDHL into the flagellar architecture. (<b>D</b>) Change in YFP::<i>Tb</i>GAPDHL localization in Tb<i>CaM</i> RNAi mutants: following RNAi induction and failure of PFR assembly YFP::<i>Tb</i>GAPDHL co-localizes with aggregates containing PFR2 protein (detected by indirect immunofluorescence with monoclonal antibody L8C4). (<b>E</b>) PFR localization of GFP::<i>Tb</i>GAPDHL is retained in <i>snl-2</i> RNAi mutants; detergent extracted cytoskeletons were also stained for indirect immunofluorescence with L13D6 to highlight failure to incorporate either PFR1 or PFR2, the two major PFR components, into the flagellar architecture. DIC, differential interference contrast; N, nucleus; K, kinetoplast. Scale bars denote 5 µm.</p

Flagellar localization of <i>Tb</i>PGKL.

<p>(<b>A</b>) Indirect immunofluorescence using monoclonal antibody BB2 reveals axonemal localization of Ty::<i>Tb</i>PGKL in detergent-extracted procyclic <i>T. brucei</i> cytoskeletons. Cytoskeletons were stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect mitochondrial (kinetoplast, K) and nuclear (N) DNA. The inset shows how the indirect immunofluorescence signal extends close to the kinetoplast, consistent with axoneme association. Scale bar denotes 5 µm. (<b>B</b>) Immunoblot analysis of detergent- and NaCl-extracted flagella isolated from procyclic cells expressing Ty::<i>Tb</i>PGK-like protein using BB2 detects a single band of the expected molecular mass.</p

Surface representation of TbGAPDHL with substrates.

<p>The representation is colored according to sequence conservation (blue-red, low to high) by Consurf. The catalytic Cys residue, neighboring residues (white sticks) and bound ligands (covalently attached glyceraldehyde-3-phosphate and NAD; ball and stick) from superimposed <i>G. stearothermophilus</i> GAPDH (PDB code 3cmc) are shown, with H-bonds illustrated as dotted lines. Corresponding residues in the TbGAPDHL model (purple sticks) are functionally incapable. Residues are labelled as template/model.</p

Supplementary Figure 1. Localisation of YFP::TbOFD1 in procyclic T. brucei; Supplementary Figure 2. Orthology of Tb927.10.3000 and HsOFD1; Supplementary Figure 3.Penetrance of TbFOP RNAi from A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Localisation of Tb927.10.3000 gene product in procyclic T. brucei. (A-B) YFP::Tb927.10.3000 is present at mature basal bodies throughout procyclic cell cycle. Monoclonal antibody YL1/2 detected tyrosinated α-tubulin prominent on subpellicular microtubules at the posterior pole of whole cells and TbRP2 (Reference 4 in the main text) at the mature basal body. Scale bars in all main panels indicate 5 µm and in the inset panels 1 µm.; Orthology of Tb927.10.3000 and HsOFD1. (A) Cartoon representation of human and T. brucei OFD1 proteins, showing insertions necessary to achieve maximal alignment of amino acid sequences. (B) Amino acid alignment of Homo sapiens (Accession number AC003037.1) and T. brucei (Tb927.10.3000) OFD1. (C) Effect of Tb927.10.3000 RNAi induction on trypanosome growth (triangles; solid line) compared to RNAi non-induced controls (diamonds; dashed line); immunoblotting with monoclonal antibody BB2 (detecting an N-terminal Ty-epitope) indicated depletion of YFP::Tb927.10.3000 post-RNAi induction. Polyclonal rabbit sera detecting trypanosome adenylate kinase isoform F (Ginger ML et al. 2005 J. Biol. Chem. 280, 11781-9 doi: 10.1074/jbc.M413821200) was used as a loading control. (D) Preliminary analysis of cell morphology following Tb927.10.3000 RNAi induction: cells were scored for normal morphology versus assembly of an abnormal, short flagellum (at 24 h post-RNAi induction n = 51 ‘non-induced’ cells / n = 67 ‘induced’ cells; 48 h post-induction n = 41 ‘non-induced’ / n = 75 ‘induced’; 72 h post-induction n = 39 ‘non-induced’ / n = 95 ‘induced’; 96 h post-induction n = 25 ‘non-induced’ / n = 123 ‘induced’). (E-M) Electron and fluorescence microscopy analysis of cell morphology: E-G, TEM analysis illustrating in short flagella the accumulation of electron dense material, potentially including unassembled PFR components, around normal 9+2 axoneme architecture; H-J, SEM analysis illustrating the short flagellum phenotype of Tb927.10.3000 RNAi mutants; K-L, fluorescence microscopy illustrating mixed ‘short flagellum’ and ‘short cell’ phenotypes in Tb927.10.3000 RNAi mutants (96 h post-RNAi induction); M, normal cell morphologies in Tb927.10.3000 cells not induced for RNAi against Tb927.10.3000. In K-M, the PFR is immunolabelled with monoclonal antibody L8C4 (red); DAPI (blue) was used to stain nuclear and kinetoplast DNA. Assessment of OFD1 candidature. Mature basal body localisation of YFP::Tb927.10.3000 is analogous to mature centriole localisation of human OFD1 (Singla V et al. (2010) Dev. Cell 18, 410-24 doi: 10.1016/j.devcel.2009.12.022). The short flagellum phenotype of the RNAi mutant resembled published T. brucei intraflagellar transport (IFT) RNAi phenotypes: the short flagellum phenotype of Tb927.10.3000 RNAi mutants is more similar in presentation to the phenotype arising from defective retrograde IFT than it is to defective anterograde IFT mutants, which fail to elongate an axoneme beyond the transition zone, and thus fail to build flagella (Absalon S et al. (2008) Mol. Biol. Cell 19, 929-44 doi: 10.1091/mbc.E07-08-0749; Davidge J et al. (2006) J. Cell Sci. 119, 3935-43 doi: 10.1242/jcs.03203). Flagellar membrane elongation or ‘flagellar sleeve’, seen in some T. brucei IFT mutants (Davidge et al. 2006), was also evident in SEM micrographs of Tb927.10.3000 RNAi-induced mutants. Loss of YFP::Tb927.10.3000 beneath the threshold of detection by immunoblot was evident 24 h post-RNAi induction, but presentation of the short flagellum phenotype was never evident across all cells in RNAi-induced cultures even by 96 h post-induction. We interpret such partial presentation of morphological phenotype, in contrast to the depletion of YFP::Tb927.10.3000 beneath a threshold level of detection, coupled to literature reports of OFD1 function in mammals, including a requirement in murine embryonic stem cells for OFD1 in formation of centriole distal-end appendages and IFT88 recruitment, to suggest that protein encoded by Tb927.10.3000 provides regulatory or indirect function(s) in IFT, rather than being a core part of the IFT machinery. In summary, candidature of Tb927.10.3000 as an OFD1 ortholog based only on amino acid sequence alignment with HsOFD1 is equivocal, but coupled to comprehensive preliminary RNAi phenotype analysis evidence for OFD1 candidature is persuasive.; Penetrance of TbFOPL RNAi. Representative fields of view for whole cells show rapid loss of normal cell morphology and aberration of normal PFR biogenesis in populations at 24 (A-C) and 48 (D-F) h post-RNAi induction. Scale bars represent 10 μm

Supplementary Figure 1. Localisation of YFP::TbOFD1 in procyclic T. brucei; Supplementary Figure 2. Orthology of Tb927.10.3000 and HsOFD1; Supplementary Figure 3.Penetrance of TbFOP RNAi from A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Localisation of Tb927.10.3000 gene product in procyclic T. brucei. (A-B) YFP::Tb927.10.3000 is present at mature basal bodies throughout procyclic cell cycle. Monoclonal antibody YL1/2 detected tyrosinated α-tubulin prominent on subpellicular microtubules at the posterior pole of whole cells and TbRP2 (Reference 4 in the main text) at the mature basal body. Scale bars in all main panels indicate 5 µm and in the inset panels 1 µm.; Orthology of Tb927.10.3000 and HsOFD1. (A) Cartoon representation of human and T. brucei OFD1 proteins, showing insertions necessary to achieve maximal alignment of amino acid sequences. (B) Amino acid alignment of Homo sapiens (Accession number AC003037.1) and T. brucei (Tb927.10.3000) OFD1. (C) Effect of Tb927.10.3000 RNAi induction on trypanosome growth (triangles; solid line) compared to RNAi non-induced controls (diamonds; dashed line); immunoblotting with monoclonal antibody BB2 (detecting an N-terminal Ty-epitope) indicated depletion of YFP::Tb927.10.3000 post-RNAi induction. Polyclonal rabbit sera detecting trypanosome adenylate kinase isoform F (Ginger ML et al. 2005 J. Biol. Chem. 280, 11781-9 doi: 10.1074/jbc.M413821200) was used as a loading control. (D) Preliminary analysis of cell morphology following Tb927.10.3000 RNAi induction: cells were scored for normal morphology versus assembly of an abnormal, short flagellum (at 24 h post-RNAi induction n = 51 ‘non-induced’ cells / n = 67 ‘induced’ cells; 48 h post-induction n = 41 ‘non-induced’ / n = 75 ‘induced’; 72 h post-induction n = 39 ‘non-induced’ / n = 95 ‘induced’; 96 h post-induction n = 25 ‘non-induced’ / n = 123 ‘induced’). (E-M) Electron and fluorescence microscopy analysis of cell morphology: E-G, TEM analysis illustrating in short flagella the accumulation of electron dense material, potentially including unassembled PFR components, around normal 9+2 axoneme architecture; H-J, SEM analysis illustrating the short flagellum phenotype of Tb927.10.3000 RNAi mutants; K-L, fluorescence microscopy illustrating mixed ‘short flagellum’ and ‘short cell’ phenotypes in Tb927.10.3000 RNAi mutants (96 h post-RNAi induction); M, normal cell morphologies in Tb927.10.3000 cells not induced for RNAi against Tb927.10.3000. In K-M, the PFR is immunolabelled with monoclonal antibody L8C4 (red); DAPI (blue) was used to stain nuclear and kinetoplast DNA. Assessment of OFD1 candidature. Mature basal body localisation of YFP::Tb927.10.3000 is analogous to mature centriole localisation of human OFD1 (Singla V et al. (2010) Dev. Cell 18, 410-24 doi: 10.1016/j.devcel.2009.12.022). The short flagellum phenotype of the RNAi mutant resembled published T. brucei intraflagellar transport (IFT) RNAi phenotypes: the short flagellum phenotype of Tb927.10.3000 RNAi mutants is more similar in presentation to the phenotype arising from defective retrograde IFT than it is to defective anterograde IFT mutants, which fail to elongate an axoneme beyond the transition zone, and thus fail to build flagella (Absalon S et al. (2008) Mol. Biol. Cell 19, 929-44 doi: 10.1091/mbc.E07-08-0749; Davidge J et al. (2006) J. Cell Sci. 119, 3935-43 doi: 10.1242/jcs.03203). Flagellar membrane elongation or ‘flagellar sleeve’, seen in some T. brucei IFT mutants (Davidge et al. 2006), was also evident in SEM micrographs of Tb927.10.3000 RNAi-induced mutants. Loss of YFP::Tb927.10.3000 beneath the threshold of detection by immunoblot was evident 24 h post-RNAi induction, but presentation of the short flagellum phenotype was never evident across all cells in RNAi-induced cultures even by 96 h post-induction. We interpret such partial presentation of morphological phenotype, in contrast to the depletion of YFP::Tb927.10.3000 beneath a threshold level of detection, coupled to literature reports of OFD1 function in mammals, including a requirement in murine embryonic stem cells for OFD1 in formation of centriole distal-end appendages and IFT88 recruitment, to suggest that protein encoded by Tb927.10.3000 provides regulatory or indirect function(s) in IFT, rather than being a core part of the IFT machinery. In summary, candidature of Tb927.10.3000 as an OFD1 ortholog based only on amino acid sequence alignment with HsOFD1 is equivocal, but coupled to comprehensive preliminary RNAi phenotype analysis evidence for OFD1 candidature is persuasive.; Penetrance of TbFOPL RNAi. Representative fields of view for whole cells show rapid loss of normal cell morphology and aberration of normal PFR biogenesis in populations at 24 (A-C) and 48 (D-F) h post-RNAi induction. Scale bars represent 10 μm

Sequence alignment of trypanosomatid GAPDHL proteins with authentic GAPDH.

<p>The alignment was built using MUSCLE and sequences are named with species abbreviations (Tb =  <i>T. brucei</i>, Tc =  <i>T. cruzi</i>, Lm =  <i>L. major</i>, Bs =  <i>B. saltans</i>, Gt =  <i>Geobacillus stearothermophilus</i>, Mb =  <i>Mycobacterium bovis</i>, Hs =  <i>Homo sapiens</i>, Ce =  <i>Caenorhabditis elegans</i>, Mj =  <i>Methanocaldococcus jannaschii</i>, Pt =  <i>Picrophilus torridus</i>) followed by a locus code (kinetoplastid sequences) or UniProt accession. For kinetoplastid sequences, cGAPDH indicates the cytosolic isoform and gGAPDH the glycosomal enzyme. Only one each of the tandem copies of gGAPDH is shown for each trypanosomatid. Residues mentioned in the text are highlighted as white on purple.</p

Range and mean percentage amino acid identities between GAPDH(-like) groups in trypanosomatids.

<p>Each group contains sequences (eliminating tandem duplicates) from <i>T. brucei, T. cruzi, T. vivax, L. braziliensis, L. mexicana, L. major, L. infantum, L. tarentolae</i>, and <i>Endotrypanum monterogeii</i>.</p