Identification of IFT-related Genes in Trypanosomes
To identify trypanosome genes participating in flagellum formation, we first searched the T. brucei
genome for genes encoding proteins known to be components of purified IFT particles in Chlamydomonas
(Cole et al., 1998
). At the time this study was initiated, the sequences of eight genes encoding IFT proteins were available: IFT122
(complex A) and IFT20
(complex B) (C. elegans
and Drosophila melanogaster
gene nomenclature is given in ). The corresponding trypanosome genes are all present in the T. brucei
genome database (Kohl et al., 2003
; Briggs et al., 2004a
; Berriman et al., 2005
; Absalon et al., 2007
) and exhibit 21–55% overall identity with orthologues from various species (), a value in the same range as what was found for axonemal proteins (Branche et al., 2006
; Ralston et al., 2006
; Baron et al., 2007
). In addition, the gene encoding the IFT dynein heavy chain previously identified and functionally characterized in our laboratory was also included in this study, hence providing a total of nine IFT-related genes.
Next, we searched to identify novel genes encoding proteins possibly involved in the IFT process but that were not purified with IFT particles. Because the only flagella of Plasmodium
are found in the male gametes and they are assembled in the cytoplasm, IFT
genes are absent from the genome of this group of species. By contrast, genes encoding proteins of the dynein arms or central pair components of the axoneme are conserved (Avidor-Reiss et al., 2004
; Kohl and Bastin, 2005
). This provides the opportunity to compare the genomes of Plasmodium
spp. with those of other ciliated species and to discriminate genes encoding proteins involved in IFT from genes encoding structural components of the flagellum. A similar approach was successfully used to identify genes encoding flagellar proteins involved in beating by comparison of species with motile or nonmotile flagella (Baron et al., 2007
). Comparison of genomes from species assembling their flagella with or without IFT indicated that at least 27 genes are conserved in species assembling their flagella by IFT (Avidor-Reiss et al., 2004
). We selected five of these genes that are conserved in T. brucei
for functional analysis and termed them PIFT
genes (). The PIFTA1 protein presents coiled-coil domains, whereas the PIFTB2 and PIFTC3 protein contain TPR domains, a feature shared with IFT88 and IFT139 (Cole, 2003
). PIFTD4 and PIFTF6 both contain two WD-40 domains at their amino terminal region and a TPR motif toward the center of their sequence. PIFTE5 exhibits a different structure and will be reported elsewhere. The five trypanosome PIFT
genes are the homologues of the recently identified DYF-3
, and DYF-2
, respectively, known to be linked to the formation of sensory cilia in C. elegans
(Starich et al., 1995
; Blacque et al., 2005
; Murayama et al., 2005
; Bell et al., 2006
; Efimenko et al., 2006
). PIFTD4 and PIFTF6 are the homologues of the OSEG4 and OSEG6 proteins that localize to the Outer SEGment of sensory cilia in Drosophila
(Avidor-Reiss et al., 2004
). Although these proteins were not detected in purified IFT particles, they are present in the membrane + matrix fraction of Chlamydomonas
flagella (Pazour et al., 2005
). The only exceptions are the IFT122 and the PIFTC3 proteins that were not detected in that analysis ().
Localization of Endogenous and Tagged IFT Proteins in Mature and Elongating Flagella
To determine the location of IFT proteins in trypanosomes, mouse polyclonal antibodies were raised against a classic IFT marker, the IFT172 protein. It has been localized to the flagellum matrix and to the basal bodies of Chlamydomonas
where it is required for flagellum assembly (Pedersen et al., 2005
) and in C. elegans
(Bell et al., 2006
). It is also involved in hedgehog signaling in mouse (Huangfu et al., 2003
). A fragment of the trypanosome IFT172
gene was expressed in E. coli
to produce a fusion protein with GST for injection in mice. In a second approach, the genes encoding IFT20 or IFT52 were selected for fusion to a protein A tag (Estevez et al., 2001
) or to GFP, respectively. IFT20 and IFT52 are proteins belonging to the B complex that have been well characterized in Chlamydomonas
(Deane et al., 2001
), C. elegans
(Collet et al., 1998
), or in mammalian cells (Follit et al., 2006
). The expression of the fusion proteins is controlled by a tetracycline-inducible promoter, and it is activated by addition of tetracycline to the culture medium. Western blot analysis with an anti-protein A antibody or with an anti-GFP antibody confirmed that the IFT20-tagged protein and the GFP::IFT52 protein are expressed upon addition of tetracycline and that they show an electrophoretic motility corresponding to the expected size of the fusion protein (Supplemental Figure S1, A and B).
Indirect immunofluorescence assay (IFA) with anti-IFT172 antibodies produced a defined pattern on trypanosomes fixed in methanol: a strong signal is observed at the basal body region, and a succession of closely spaced spots is found all along the length of the flagellum until its distal tip (A). The same pattern was observed in cells possessing two flagella, and no visible difference in signal could be detected between the new (no matter its length) and the old flagellum (see, for example, the cell at the bottom left of A). The same antiserum used on PFA-fixed trypanosomes stained the flagellar compartment but not the basal body (data not shown), possibly due to a more difficult access as noted previously for IFT20 in mammalian cells (Follit et al., 2006
). IFT20 fused to a protein A tag also localized to the flagellum and to the basal body, with a weak signal on the cell body (B). Double staining with markers of various organelles (endoplasmic reticulum, lysosomes, mitochondria) failed to reveal a specific association, indicating that the protein is present in the cytosol, possibly as a consequence of overexpression (Supplemental Figure S1, C and D; data not shown). Finally, observation of live cells expressing GFP::IFT52 also demonstrated localization to the basal body and the probasal body and to the flagellar compartment of both old and new flagellum, with a more pronounced localization at the proximal part of the flagellum (data not shown). This pattern was preserved after methanol fixation, although it became less intense (C). Staining with an anti-GFP antibody confirmed this localization (Supplemental Figure S1, F and G).
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 (more ...)
To further investigate IFT proteins, trypanosomes were treated with 0.1% Nonidet-P40 before fixation. Detergent addition removes the cell body and flagellum membranes but the subpellicular corset of microtubules, the axoneme, the PFR and the basal body complex remain intact (Sherwin and Gull, 1989
). Detergent treatment abolishes most of the flagellum staining for IFT172, tagged IFT20, and GFP::IFT52, but a clear signal remains present at the basal body region in both methanol- and PFA-fixed cells (, D and F). This indicates that a pool of IFT172 with possibly different biochemical characteristics is present around the basal bodies, as recently suggested for Bardet–Biedl syndrome (BBS) proteins (Nachury et al., 2007
To further define the positioning of IFT proteins, GFP::IFT52-expressing cells were stained with several markers of the basal body (, A–D). Double labeling with MAb22, a mAb marker of the proximal region of both mature and probasal bodies (Pradel et al., 2006
; Absalon et al., 2007
), showed that GFP::IFT52 was found in a more apical position (A), a feature that was reproduced for tagged IFT20 (Supplemental Figure S1E). The antibody against trypanosome basal body component (TBBC) (Dilbeck et al., 1999
), a protein found exclusively on the mature basal body that hence produces a single spot in IFA (Absalon and Bastin, unpublished data) showed that GFP::IFT52 was present in an even more apical position (B). Costaining was also performed with two antibodies against centrins that localize to mature and probasal bodies, and to a “bilobed” structure found close to the Golgi apparatus (He et al., 2005
; Selvapandiyan et al., 2007
). The mAb 20H5 recognizes numerous centrin proteins from different organisms, and in detergent-extracted trypanosomes it was found to light up the flagellum, the mature and the probasal body, and a structure close to the flagellar pocket and the Golgi apparatus (C). A similar pattern was obtained with the antiserum raised against centrin 1 from the related organism Leishmania donovani
, except that the flagellum and basal body signals were weaker (D). In both cases, GFP::IFT52 was found to merge with the centrin signal at the mature basal body. The relative position of IFT proteins themselves was investigated upon staining of GFP::IFT52-expressing cells with the anti-IFT172 antiserum, revealing a close but separate location for the two proteins. GFP::IFT52 was found to be in a more apical location compared with IFT172 (Supplemental Figure S2). Double staining of tagged IFT20-expressing cells with the anti-protein A antibody and the anti-IFT172 antiserum showed that these two proteins colocalized at the basal body region (Supplemental Figure S2).
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), (more ...)
In conclusion, three different IFT proteins are present along the length of the axoneme where they are sensitive to detergent, and at the mature basal body and frequently at the probasal body where they are resistant to detergent. Importantly, they localize to both old and new flagella, indicating that IFT could operate in mature and elongating flagella.
Movement of GFP::IFT52 Protein in Trypanosome Flagella
Presence of various IFT proteins in both old and new flagella does not necessarily prove that these proteins are trafficking in this compartment. We therefore examined the behavior of the GFP::IFT52 fusion protein in live trypanosomes. Agarose was added to the medium to restrict cell movement, and direct GFP fluorescent signals were monitored by videomicroscopy. This showed unambiguous movement of fluorescent particles in flagella, providing the first actual evidence for intraflagellar transport in trypanosomes (Supplemental Videos 1 and 2; still images of Movie 1 are presented at E). Intraflagellar movement was detected in both old and new flagella, but it was more difficult to quantify on the new flagellum because this flagellum is regularly found on top of the cell body, which tends to obliterate the fluorescent signal. IFT was best visualized on the distal end of the mature flagellum (Supplemental Movies S1 and S2), but it was also detected in the new flagellum when it lied on the side of the cell (Supplemental Movie S1). Anterograde events were more frequent and easier to observe (Supplemental Movies S1 and S2), but a few retrograde events could also be detected (Supplemental Movie S2). Rate of anterograde IFT was measured at ~3 μm/s. Retrograde transport was faster, but it could not be estimated accurately due to its low frequency and to the weak GFP signal. These data demonstrate that IFT is indeed active in trypanosomes and operates in both mature and assembling flagella.
IFT-like Particles in the Trypanosome Flagellum
, IFT particles can be recognized on flagellum sections as electron-dense granules found between the membrane and the axoneme (Kozminski et al., 1993
). Occasional viewing of such particles has been previously reported in T. brucei
, but detailed analysis is lacking (Bastin et al., 2000b
). We examined a large number of cross sections of flagella from wild-type and several RNAi motility mutants (Branche et al., 2006
) and noticed the frequent presence of granule-like structures with a diameter of 20–30 nm (, A–F, and Supplemental Figure S3). Usually, only one particle is visible per section (, A and C) but in rare instances (<5%), several particles can be seen on the same section (B). Remarkably, particles show a very specific location relative to the axoneme doublets (D): they are either associated to doublets 3–4 (A) or 7–8 (C), but they are never found next to doublets 5–6, doublet 9, and rarely close to doublets 1 and 2 (D). Longitudinal sections showed that the particles look like flat rafts with one side that is very close to the microtubules, and the other side that seems almost in contact with the flagellar membrane (E). Presence of the IFT-like particles seems to position the flagellar membrane more closely toward the axonemal microtubules (E).
Figure 4. IFT particles in flagella of trypanosomes. (A–C) Cross sections through the flagella of wild-type (WT) trypanosomes (A and B) or PF16RNAi (C) cells induced for 3 d. The presence of the PFR attached to the axoneme allows for unambiguous identification (more ...)
We also looked at flagellum sections of RNAi mutants where expression of two proteins of the central pair (PF16 or PF20) had been separately silenced, leading to inhibition of motility but without interfering with flagellum construction (Branche et al., 2006
; Ralston et al., 2006
). Similar particles are visible in these flagella (C) although at a slightly lower frequency: 31% of sections for PF16RNAi
cells and 24% for PF20RNAi
cells instead of 57% for wild-type (L). Nevertheless, they all show the preferential association to the same doublets as was found in wild-type cells (D).
Because the new flagellum is always found in the same position relative to the old flagellum (Briggs et al., 2004b
), it can be identified in the few sections where both flagella are clearly visible. This revealed that both flagella contain these particles (a clear example for the old flagellum is shown at F). IFT-like particles are never recognized in the transition zone of the basal body, although some amorphous, relatively electron-dense, material is visible between the microtubule doublets and the membrane (G). This was clearly different from the inside of the axoneme shaft that seems much more translucent (G). The central pair and the dynein arms are already visible in flagellum sections close to the top of the flagellar pocket (as confirmed by the short diameter of the flagellar pocket lumen). The PFR is not yet present but a large amount of material is found around the microtubule doublets, sometimes resembling the granules identified above (, H–I).
Detergent-treatment suppresses most of the IFT172, tagged IFT20 or GFP::IFT52 signal at the flagellum. To relate this result with granules seen by electron microscopy, wild-type, PF16RNAi
, or PF20RNAi
cells were treated with 1% Nonidet P-40. This procedure removes the cell membrane, but it ensures an excellent conservation of the whole trypanosome cytoskeleton for electron microscopy analysis, including the axoneme and the PFR (Sherwin and Gull, 1989
). In these conditions, the frequency of IFT-like particles drops, with <10% of samples showing a possible remnant at the expected position on doublets 3–4 or 7–8 (, J–L). This result shows that these granules display the same characteristics as the three IFT proteins studied above.
IFT Genes Are Necessary for Flagellum Construction
Expression of the trypanosome genes IFT122
(complex A) and IFT20
(complex B) was individually silenced by inducible RNAi in trypanosomes, and knockdown efficiency was controlled at the RNA level by semiquantitative RT-PCR (Supplemental Figure S4). In all cases, RNAi silencing led to the inhibition of new flagellum formation and to the emergence of apparently nonflagellated trypanosomes (, A and B). However, the old flagellum is not depolymerized and remains present. Analysis of cells fixed at different stages of RNAi silencing followed by IFA staining using a mAb recognizing a major PFR protein (, A and B) showed a mixture of different cell types: cells retaining the old flagellum, cells with a shorter flagellum and cells without PFR signal, that could correspond to cells with very short flagella missing the PFR or to nonflagellated cells (see below). Cells with shorter flagella exhibited a reduced cell size, with those without a visible flagellum being the shortest (, A and B), in agreement with the central role of the flagellum in the definition of cell size (Kohl et al., 2003
). Trypanosomes are difficult to synchronize reliably, meaning that RNAi induction is likely to have different effects according to the position of a cell in its cycle. Therefore, it is important to examine carefully cells throughout the induction. IFA with the anti-PFR was first used to score proportions of positive and negative cells during the course of RNAi silencing of IFT
and genes (, D and E). Significant differences were noted in the rate of emergence of nonflagellated cells. In complex A proteins, knockdown of IFT140 leads to faster emergence of nonflagellated cells compared with IFT122 (D). Differences are also observed in the knockdown of RNA-encoding complex B proteins. For example, it takes only 30 h to obtain 50% of nonflagellated cells during silencing of IFT172
, whereas it takes an extra 40 h to generate a similar result for IFT52
knockdown (E). Hence, these differences in kinetics are not linked to genes encoding members of a specific complex. Inhibition of any of the IFT
genes invariably leads to a growth arrest whose timing is correlated with the kinetics of emergence of nonflagellated cells (, D and G and E and H, for proteins of complex A and B, respectively). For example, in complex A members, silencing of IFT140 rapidly produces a large number of nonflagellated cells and leads to a more premature growth arrest compared with IFT122RNAi
grown and induced in the same conditions (G).
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 (more ...)
These differences in kinetics could reflect variable efficiency of RNA destruction or different rates of protein turnover (or both). RT-PCR analysis was performed on total RNA extracted from cell lines noninduced or induced for up to 5 d. Abundance of each target RNA was normalized with a control unrelated mRNA (Supplemental Figure S4). Kinetics of RNA silencing is very reproducible for all RNAi cell lines investigated: a potent silencing is observed 2 d after induction and a slight increase in the amount of the target RNA is noted the following days. This reproduces kinetic patterns observed for other RNAi cell lines developed in separate experiments (Durand-Dubief, Absalon, and Bastin, unpublished data). In summary, the differences in kinetics of emergence of nonflagellated cells during the course of RNAi are not due to a variable efficiency of RNA degradation, but they are likely to reflect differences at the protein level.
Distinct Phenotype upon Silencing of Genes Required for Anterograde or Retrograde Transport
and C. elegans
, inhibition of proteins associated to anterograde transport (IFT B and kinesin II complexes) leads to complete or severe inhibition of flagella or cilia formation. By contrast, blocking of retrograde transport (IFTA and dynein complexes) produces short cilia or flagella, filled with IFT material that can penetrate the flagellar compartment but cannot be recycled to the base (Cole, 2003
). We analyzed IFT88RNAi
(mutant of a complex B protein) and IFT140RNAi
(mutant of a complex A protein) induced cells by double staining with the anti-IFT172 antiserum as a marker of the IFT particles and with the MAb25 antibody as a marker of the axoneme (, A and C, and E). In IFT88RNAi
, the majority of short cells did not exhibit a flagellum (no MAb25 signal) and only showed a weak signal for IFT172, indicating that absence of IFT88 severely blocks flagellum formation and leads to a reduction or a dispersion of IFT172 (A, stars, and C). This result was supported by scanning electron microscopy that revealed that most short cells did not exhibit a flagellum. In the rare cases where a flagellum could be detected, it often seemed short (<1 μm) and thin (D). Cells that retained the old flagellum were still positive for both the MAb25 axoneme marker and the anti-IFT172, no matter the length of the flagellum (A, arrows for long flagellum and arrowheads for flagella of intermediate lengths). A very different result was obtained for the IFT140RNAi
cells stained with the same antibodies (, B and E). Numerous short cells were seen with a short line as MAb25 signal, indicating formation of a short axoneme, and with a very intense signal for IFT172 (, B and E). The presence of short flagella filled with IFT172 proteins, producing very bright spots of fluorescence, increased rapidly during the course of RNAi silencing, whereas it was virtually never detected in the case of IFT88RNAi
(G). Scanning electron microscopy demonstrated the presence of cells with short flagella (1–3 μm) with large dilations (F), that very likely results from the accumulation of IFT proteins. However, cells that retained an old flagellum of normal length still exhibited the usual MAb25 and anti-IFT172 signals (B, arrows). By contrast, cells that possessed a flagellum of intermediate length (~5 μm) showed normal MAb25 signal but much increased abundance of IFT172 protein (B, arrowhead).
Figure 6. IFT proteins from complex A and B are required for flagellum formation but differ in function. (A and B) Fields of IFT88RNAi (A) or IFT140RNAi (B) cells induced for 3 d stained with the anti-IFT172 antiserum (green) and with the MAb25 axoneme marker (red). (more ...)
Although construction of the new flagellum is inhibited or strongly reduced, the old flagellum remains present and is still motile. We examined IFT140RNAi cultures at early stages of RNAi, with special emphasis on cells that were binucleated, so at late stages of their cell cycle (H). The mature flagellum was normal and exhibited the expected signal for the anti-IFT172 antiserum whereas the new flagellum was very short and filled with IFT172 protein. This shows that the old and the new flagellum are independent from each other and despite the arrest of new IFT140 production, IFT140 protein present in the old flagellum is still functional. The ultrastructure of the old flagellum of IFT88RNAi and IFT172RNAi (anterograde transport mutants) and DHC1bRNAi (retrograde transport mutant) was examined by transmission electron microscopy. Induction times were selected in such a way that cells were at a stage when they had stopped constructing a new flagellum, so that almost all flagellar sections should correspond to the old flagellum. This was carried out on whole cells and detergent-extracted cytoskeletons (Supplemental Figure S3). Sections through flagella were less frequent on induced cells compared with wild-type or noninduced controls, as expected from the drop in the proportion of flagellated cells upon RNAi induction. However, the ultrastructure of the axoneme seemed intact: the nine doublets microtubules, the central pair, the dynein arms and radial spokes were all present (Supplemental Figure S3). Their flagellar pocket also seemed structurally normal, as well as the flagellum adhesion zone (Supplemental Figure S3). Importantly, IFT particles are still visible on the old flagellum (Supplemental Figure S3). We observed IFT-like particles on six of 19 such profiles, i.e., a proportion similar to that observed for populations of cells that build their flagellum normally, indicating that IFT particles remain present in the old flagellum. This is confirmed by IFA with the anti-IFT172 that still stains the old flagellum of IFT140RNAi (H), IFT88RNAi and DHC1bRNAi (data not shown) cells induced for 2 or 3 d, indicating a slow turnover of IFT proteins in mature flagella.
Transmission electron microscopy was used to examine the new flagellum in control wild-type cells or IFT88RNAi
cells (). The basal body is rooted within the cell body, whereas the transition zone is covered by the flagellum membrane and localized in the lumen of the flagellar pocket of wild-type cells (A). The flagellar pocket exhibits a typical asymmetric structure with the larger lumen side found at the anterior end. When a new flagellum is assembled, it is made in the same flagellar pocket (Grassé, 1961
) found on the large, anterior, side of the lumen (B, arrow). The transition zone is clearly recognizable and a mass of amorphous material is found at the distal growing end (, B and C), wrapped by the flagellum membrane that sometimes seems distorted (B). The basal bodies of such flagella are “bald”, i.e., axoneme microtubules have not yet elongated. These basal bodies seem shorter, suggesting that they have not fully matured yet. This was encountered in five of the 72 analyzed sections of the flagellar pocket (7%) (). The new flagellum grows and axoneme microtubules are now visible in continuity with those of the transition zone. Nevertheless, this new flagellum remains in the same flagellar pocket as the old flagellum (D). In nonsynchronous cultures of wild-trypanosomes, 16% of flagellar pockets contain two flagella (n = 72). Finally, two individual flagellar pockets are visible, each containing a single flagellum (E).
Figure 7. Formation of the new flagellum in wild type cells and its inhibition in IFTRNAi cell lines. Sections through the flagellar pocket of WT (A–E) or IFT172RNAi cells induced for 48 h (F) or of IFT88RNAi (G) or DHC1bRNAi (H and I) induced for 72 h. (more ...)
Effects of RNAi silencing of IFT genes on axonemal microtubule elongation
Ultrastructure of the basal body and the flagellum was analyzed in IFT88RNAi, IFT172RNAi (mutants of anterograde transport), and DHC1bRNAi (mutant of retrograde transport) cells at induction times where assembly of the new flagellum is drastically reduced (, F–I). Longitudinal sections through basal bodies were grouped in three categories: those where axonemal microtubules are visible in direct continuity of the transition zone and seem normal, those where axonemal microtubules are present but interrupted, and those where only a bald basal body is visible (). In all IFTRNAi mutants analyzed, the frequency of bald basal bodies rose significantly reaching close to 80% in the IFT88RNAi cell line (). Some material is frequently detected on top of the basal body and looks like flagellar membrane extension (see below), but it does not resemble to what was observed for wild-type flagella (compare F with B and C). The proximal region of all basal bodies still exhibits triplet microtubules, and the transition zone looks apparently normal. Nevertheless, these basal bodies seem slightly shorter, like the basal bodies of short new flagella in wild-type cells (, B and C). These data demonstrate that axoneme formation is strongly inhibited in the absence of a protein of the IFT B complex. In the case of DHC1bRNAi cells, about half of the sections revealed the presence of a bald basal body but other situations were encountered. Large accumulation of electron-dense material resembling IFT particles was observed (H). Moreover, several sections showed the presence of short microtubules and some material trapped between this axoneme and the tip of the flagellar membrane (I). In these situations, the basal body displayed a normal length. This accumulation fits with the dilation of short flagella of retrograde transport mutants observed by scanning electron microscopy and by IFA with the anti-IFT172 antiserum ().
We conclude that in the absence of retrograde transport, members of the IFT complex B can still access the flagellum and accumulate there as they cannot be recycled due to the absence of retrograde activity. This would allow the formation of short and dilated flagella. Conversely, in the absence of anterograde transport, entry of IFT protein in the flagellum is restricted and flagellum formation is more strongly inhibited.
PIFT Genes Are Required for Flagellum Construction and Can Be Classified as Involved in Anterograde or Retrograde Transport
Expression of PIFT genes was knock downed by RNAi, it was monitored in parallel to the mutants of conventional IFT genes, revealing that all five selected PIFT genes were required for flagellum formation (, C and F). Significant differences were also observed in the emergence of nonflagellated cells (F), with fast phenotypes for silencing of PIFTA1, PIFTB2 or PIFTC3 (50% nonflagellated cells in 48–60 h), and the slower kinetics for PIFTF6 knockdown (50% of nonflagellated cells after 120 h of induction) and PIFTD4 (3–5% of nonflagellated cells after 120–240 h of induction; data not shown). These differences correlated with the timing of growth arrest (I; see Supplemental Figure S5, A and B, for PIFTF6RNAi), as observed in the case of silencing conventional IFT genes (, G and H).
To evaluate the participation of these novel genes in IFT, IFA staining with the anti-IFT172 was performed on the five PIFTRNAi cell lines ( and Supplemental Figure S6). Flagellum presence and IFT172 signal were drastically reduced in PIFTA1RNAi, PIFTB2RNAi, and PIFTC3RNAi. In the latter case, an increase of the cytoplasmic signal was consistently observed, indicating a possible redistribution of IFT172 in the cytoplasm (B). In contrast, cells with short flagella brightly stained by the anti-IFT172 antiserum were abundant in PIFTD4RNAi and PIFTF6RNAi induced cells. Quantification of the proportion of short cells with bright, weak or no IFT172 signal was performed on all five PIFTRNAi cell lines and revealed that PIFTA1RNAi, PIFTB2RNAi and PIFTC3RNAi behave similarly to IFT88RNAi (complex B), whereas PIFTD4RNAi and PIFTF6RNAi behave similarly to IFT140RNAi (complex A) (D). Scanning electron microscopy demonstrated that when visible, short flagella of PIFTA1RNAi were smaller and thinner compared with those from PIFTD4RNAi or PIFTF6RNAi (Supplemental Figure S6). These results support the hypothesis that PIFTA1, PIFTB2, and PIFTC3 proteins are involved in anterograde transport whereas PIFTD4 and PIFTF6 are involved in retrograde transport.
Figure 8. PIFT are involved in anterograde or retrograde transport. PIFTB2RNAi (A) and PIFTC3RNAi (B) induced for 3 d and PIFTD4RNAi (C) induced for 5 d were stained with the anti-IFT172 antiserum. (D) Proportion of short cells displaying no, weak or bright signal (more ...)
Flagellar Membrane Extension Can Take Place in the Absence of IFT
While analyzing IFTRNAi
cells by scanning electron microscopy, we frequently noticed the presence of membrane extensions of various length and shape. To understand the signification of this phenotype, glutaraldehyde-fixed samples from IFT20RNAi
(complex B), IFT140RNAi
(complex A), PIFTA1RNAi
cell lines at various stages of RNAi silencing were investigated (). At least five categories of short cells could be defined and their proportion was quantified in the various RNAi cell lines analyzed (, A and B): 1) “smooth” cells with no visible flagella nor other recognizable structures (however, these could be hidden at the other face of the sample; Aa); 2) cells without flagella but with an unusual surface “depression” that might correspond to an “empty” flagellar pocket (identification is difficult without specific markers; A, b and c); 3) cells with a “filament” that is often resolved as a close succession of small “balls” or sometimes as large vesicles that emerge from a depression without recognizable flagellar structure (yellow arrowheads on the Ad); 4) short flagella (yellow arrow) alone (Ae); and 5) short flagella with a thin extension (Af). This last case could correspond to the “flagellar sleeve,” an amazing, long and thin extension of the flagellar membrane described for short flagella of the IFT80RNAi
mutant (Davidge et al., 2006
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 PIFTF6RNAi cells induced for 4 d: cells with no recognizable structures (gray code) (more ...)
As induction of RNAi silencing goes on, the proportion of cells with a short flagellum drops rapidly (B). This drop is more severe in the case of knockdown of members of the IFT complex B (IFT20 and IFT172, <25% of flagellated cells) and of PIFTA1 compared with complex A (IFT140, close to 50% of flagellated cells) or PIFTF6 (B). Cells with a flagellar sleeve were observed in all IFTRNAi
mutants, and in the DHC1bRNAi
mutant, indicating that this is a consequence of inhibition of flagellum formation rather than a phenotype specific to IFT80 (Davidge et al., 2006
) (). Examination of cells that still possess an old flagellum demonstrated that when a new short flagellum is still visible, the flagellar sleeve is present starting from the distal tip of this flagellum and connecting to the old flagellum, presumably at the position of the flagellar connector, a structure that links the tip of the new flagellum to the side of the old flagellum (Moreira-Leite et al., 2001
; Kohl et al., 2003
; Davidge et al., 2006
) (C and Supplemental Figures S5C and S6). When no new flagellum was visible, a succession of vesicle-like structures was found at the expected location of the flagellar sleeve (Supplemental Figure S6C). Transmission electron microscopy confirmed that these vesicles emanate from the flagellar pocket and adhere to the cell surface (C). These results indicate that inhibition of axoneme construction does not prevent flagellum membrane elongation. This was observed in anterograde transport mutants despite the total inhibition of axoneme assembly (F where only a bald basal body is present). These results suggest that separate pathways are acting for formation of the cytoskeletal and the membrane compartment of the flagellum (Davidge et al., 2006