Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes

Abstract

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3-4 and 7-8, suggesting the existence of specific IFT tracks. Rapid (>3 microm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.

Figure 1.

IFT and PIFT proteins investigated in this study. Top, diagram (to scale) illustrating the various IFT proteins from complex A and B and PIFT with their specific domains. Bottom, list of IFT proteins and name of their orthologues in C. reinhardtii, in C. elegans, and in D. melanogaster. The numbers in parentheses show the percentage of identity between the trypanosome and the indicated orthologous proteins. Most IFT proteins are found in the membrane + matrix fraction of Chlamydomonas proteome. MM/Axo, the number of peptides corresponding to each protein found in the membrane + matrix (MM) or the axoneme (Axo) fractions. Data are from the proteomic analysis of the C. reinhardtii flagellum (Pazour et al., 2005).

Figure 2.

Localization of IFT proteins in trypanosome flagella. (A) Wild-type cells have been fixed in methanol for 5 min and stained with a 1:200 dilution of the anti-IFT172 antiserum (green). (B) Cells expressing IFT20 fused to a protein A tag stained with the anti-protein A antibody (green) after methanol fixation. (C) Direct observation of GFP (green) after methanol fixation of GFP::IFT52- expressing cells. (D–F) Cells from the same populations as shown in A–C but that were detergent-extracted to remove the membrane before PFA (D and E) or methanol (F) fixation. Only the basal body signal and sometimes the proximal part of the flagellum remain positive. All samples were counterstained with DAPI (blue) to visualize nuclear and kinetoplast DNA. Bar, 5 μm.

Figure 3.

GFP::IFT52 is found at the flagellum and the basal body and displays IFT in old and new flagella. (A–D) GFP::IFT52-expressing cells were fixed in methanol and stained with different antibody markers of the basal body: MAb22 (A), anti-TBBC (B), 20H5 (anti-centrins, C), and an antibody raised against the L. donovani centrin 1 (D). Direct GFP fluorescence is shown in green, the other antibodies are shown in red and DAPI in blue. Insets show 2.5-fold magnification of the indicated basal body regions. (E) Still images of Supplemental Video S1. The first panel shows a phase contrast image of the cell and the others show a succession of fluorescent images with the elapsed time indicated in seconds. The arrow points at a moving IFT particle. Bar, 5 μm.

Figure 4.

IFT particles in flagella of trypanosomes. (A–C) Cross sections through the flagella of wild-type (WT) trypanosomes (A and B) or PF16^RNAi (C) cells induced for 3 d. The presence of the PFR attached to the axoneme allows for unambiguous identification of individual doublets, shown by white numbers. IFT-like particles are indicated by black arrows. (D) Position of IFT-like particles relative to axoneme doublets on cross sections of WT, PF16^RNAi, or PF20^RNAi induced cells for 3 d (n = 42, 81, and 21, respectively). (E) Longitudinal section through an IFT-like particle. Note the closer positioning of the flagellar membrane at the particle position. (F) Cross section through both the old (bottom) and the new (top) flagella from a wild-type trypanosome revealing the presence of IFT-like particles in the old flagellum. (G) Cross section through the transition zone showing the 9 + 0 arrangement but without IFT-like particles. (H and I) Cross sections of flagella of WT trypanosomes close to the top of the flagellar pocket. Central pair and dynein arms are visible, but the PFR is not yet present. Abundant particular material can be recognized. (J and K) Cross section through flagella of wild-type or PF20^RNAi cells induced for 3 d after detergent removal of the membrane. IFT particles are less abundant in detergent-treated samples but can sometimes be recognized (arrow in K). (L) Abundance of IFT particles in cross sections of flagella from whole cells or detergent-extracted cytoskeletons of wild-type (n = 42 and 41, respectively), PF16^RNAi (n = 82 and 73, respectively) or PF20^RNAi (n = 21 and 74, respectively) cells induced for 3 d.

Figure 5.

RNAi silencing of IFT and PIFT genes inhibits new flagellum formation and results in growth arrest. Cell lines were grouped as mutants of genes encoding proteins of complex A (A, D, and G), B (B, E, and H) or PIFT (C, F, and I). (A–C) Fields of cells from the indicated cell lines that had been induced for 3 d were stained with the anti-PFR2 L8C4 antibody (green) and with DAPI (blue). A mixture of nonflagellated and flagellated cells was present in all situations. (D–F) Proportion of flagellated cells left in the culture of the indicated RNAi mutants during the course of induction of RNAi silencing. (G–I) Growth curves of induced (plain lines) and noninduced (discontinuous lines) cells from the indicated RNAi mutants.

Figure 6.

IFT proteins from complex A and B are required for flagellum formation but differ in function. (A and B) Fields of IFT88^RNAi (A) or IFT140^RNAi (B) cells induced for 3 d stained with the anti-IFT172 antiserum (green) and with the MAb25 axoneme marker (red). DNA was stained with DAPI (blue). Cells with a normal-length flagellum (arrows), with a short flagellum (arrowhead), or without visible flagellum (stars) are indicated. (C and D) IFT88^RNAi induced cells have a tiny or no flagellum. (E and F) IFT140^RNAi-induced cells exhibit short and dilated flagella accumulating a large amount of IFT172 protein. (C and E) Cells induced for 3 d and stained with the anti-IFT172 antiserum (green) and with MAb25 (red). DNA was stained with DAPI (blue). (D and F) Scanning electron micrographs. Bar, 1 μm. Inset shows a twofold magnification of the indicated area. (G) Quantification of the number of cells exhibiting accumulation of IFT172 as bright spots during the course of RNAi silencing in the indicated cell lines. (H). IFT140^RNAi cell induced for 3 d with a short new flagellum and an old flagellum. Only the new flagellum is too short, dilated and accumulates IFT172 protein. Cells were stained with the anti-IFT172 antiserum (green) and with DAPI (blue). Bar, 5 μm.

Figure 7.

Formation of the new flagellum in wild type cells and its inhibition in IFT^RNAi cell lines. Sections through the flagellar pocket of WT (A–E) or IFT172^RNAi cells induced for 48 h (F) or of IFT88^RNAi (G) or DHC1b^RNAi (H and I) induced for 72 h. Notice the large amount of material only in very short flagella of wild-type cells (B and C). The arrow in B indicates the new flagellum. The new flagellum grows in the same flagellar pocket as the old flagellum (B and D) and elongates until two separate flagellar pockets are recognized (E). Bald basal bodies (F and G) were frequent in sections of induced IFT172^RNAi and IFT88^RNAi mutants whereas short flagella and accumulation of IFT material was common in DHC1b^RNAi induced cells (H and I). Bars, 500 nm except where indicated.

Figure 8.

PIFT are involved in anterograde or retrograde transport. PIFTB2^RNAi (A) and PIFTC3^RNAi (B) induced for 3 d and PIFTD4^RNAi (C) induced for 5 d were stained with the anti-IFT172 antiserum. (D) Proportion of short cells displaying no, weak or bright signal with the anti-IFT172 antiserum (n ≥ 100). Bar, 5 μm.

Figure 9.

Inhibition of IFT does not prevent flagellum membrane elongation. (A) After RNA silencing of IFT and PIFT genes, five categories of cells were defined, illustrated here in PIFTF6^RNAi cells induced for 4 d: cells with no recognizable structures (gray code) (a); cells without flagella but with a surface depression (light blue) (b); cells with tight (c) or disperse (d) rows of vesicles that emerge from a depression without recognizable flagellar structure (dark blue); short flagella alone (orange) (e); and short flagella with a flagellar sleeve (f) as described by Davidge et al. (2006) (red). Yellow arrowheads and arrows indicate cell depressions and short flagella, respectively. (B) Frequency of each cell types in the indicated cell lines (cells with flagella longer than 2 μm were not included). (C) Section through the flagellar sleeve of IFT172^RNAi cells induced for 2 d.