Fig 1.
FAZ2 null mutant Leishmania parasites grew slower and had flagellum-to-flagellum connections.
(A) Schematic of Leishmania promastigote flagellar pocket from two different angles, giving a side-on and plan view of the flagellum attachment zone (FAZ). The flagellar pocket is divided into two regions (bulbous and neck) with the microtubule quartet (MtQ) wrapping around the bulbous region of the flagellar pocket before terminating in the neck region. The different FAZ domains and proteins present in these domains are shown. (B) Light micrographs of parental, FAZ2 null mutant and FAZ2 add back cells expressing the flagellum membrane protein SMP1 tagged with eGFP-Ty (green) and the DNA is stained with Hoechst 33342 (cyan). FAZ2 add back cells were also expressing mChFP::FAZ2 (magenta). Scale bar is 5 μm. (C) Growth curve of the parental, FAZ2 null mutant and FAZ2 add back cells over a 72 h time period. The mean ± s.d. from 3 independent experiments is plotted. (D) Light micrographs of cell types observed in culture. Scale bar is 5 μm. (E) Quantitation of cell types seen in culture for parental, FAZ2 null mutant and FAZ2 add back cells. The mean ± s.d. from 3 independent experiments is plotted. For each experiment ≥91 cells were counted. (F) Cell cycle category counts for parental, FAZ2 null mutant and FAZ2 add back cells. F–flagellum, K–kinetoplast, N–nucleus, F to F–two cells connected via their flagella. The mean ± s.d. from 3 independent experiments is plotted. For each experiment ≥163 cells were counted. (G) Light micrographs of FAZ2 null mutant cells expressing SMP1::eGFP-Ty showing the phase and SMP1::eGFP-Ty channels, with an example of two FAZ2 null mutant cells connected via their flagella and of a one flagellum FAZ2 null mutant cell that had a residual structure on the flagellum near the anterior cell tip (white arrow). Scale bar is 5 μm.
Fig 2.
FAZ2 null mutants had an altered flagellar pocket shape and reduced level of flagellum attachment.
(A) Representative light microscopy image of a parental cell with Hoechst stained DNA (cyan), with kinetoplast to anterior cell tip measurement indicated. Measurement of the distance between the kinetoplast and the anterior end of the cell body for the parental, FAZ2 null mutant and FAZ2 add back cells. These measurements were done independently 3 times on at least 100 1K1N cells. The mean of each replicate is plotted as a circle with the mean and s.d. of these individual means plotted as black lines. An unpaired, two tailed t-test was used to calculate the p value. (B) Representative electron micrograph of longitudinal section through the flagellar pocket of a parental cell and FAZ2 null mutant cell. (i) represents the distance between the basal body and the flagellar pocket collar and (ii) represents the distance between the flagellar pocket collar and the anterior cell tip. Scale bar is 500 nm. (C) Measurement of i and ii highlighted in (B). Each measurement (parental n = 34, FAZ2 null mutant n = 35) from one biological replicate was plotted with the mean represented as a red line. (D) Quantitation of the different cross sectional profiles across the flagellum and cell body observed as the flagellum extends through the flagellar pocket into the flagellar pocket neck region between the parental cells (n = 39) and FAZ2 null mutant (n = 58) from one biological replicate. Electron micrographs illustrate the two profiles based on the presence or absence of the attachment between the flagellum and the cell body. Scale bar is 200 nm.
Fig 3.
FAZ2 null mutants had a shorter FAZ filament and an extension of the anterior cell tip along the flagellum.
(A) Model of flagellar pocket generated from tomogram of parental cell. The cell body membrane is blue with the flagellum membrane in grey. The microtubule quartet (red) is nucleated close to the basal bodies, then wraps around the flagellar pocket bulbous domain before extending along the flagellar pocket neck. Additional microtubules (red) extend away from the flagellar pocket into the cell on the opposite side to the microtubule quartet. FAZ filament in yellow marked by a white asterisk runs alongside the microtubule quartet in the flagellar pocket neck. Small orange spheres mark the junctional complexes attaching the flagellum to the cell body. (B) Longitudinal tomogram slice through model in (A) along dotted black line. The regular electron dense junctional complexes mediate attachment of the flagellum (white bracket). The attached side of the cell body extends further along the flagellum than the opposite side. (C) Cross-sectional tomogram slice through model in (A) along dotted black line. White asterisk marks the FAZ filament. (D) Model of flagellar pocket generated from tomogram of FAZ2 null mutant. The cell body membrane is blue with the flagellum membrane in grey. The FAZ filament in yellow is much shorter (white asterisk), whilst the microtubule quartet appeared unaffected by FAZ2 deletion. A projection of cell body extended along the flagellum. There were fewer junctional complexes (small orange spheres) with the majority found in the cell body projection (black arrowheads). (E) Longitudinal slice through model in (D) along the dotted black line. The flagellum was still connected to the cell body (white bracket) and distinct fibres connecting the cell body and flagellum membrane were seen (white arrowheads). (F) Cross-sectional slice through model in (D) along the dotted black line. (G) Enlarged image of area indicated by a dotted box in (D) showing the shorter FAZ filament (white asterisk). (H) Enlarged image of area indicated by a dotted box in (D). The cell body membrane is blue with the flagellum membrane in grey and the sub-pellicular microtubules are in red. Junctional complexes are represented by orange spheres. (I) Longitudinal slice through (H) showing the disorganised junctional complexes in the cell body extension (white arrows) with the sub-pellicular microtubules marked by the white arrowhead. Scale bar is 200 nm.
Fig 4.
Deletion of FAZ2 affected the localisation of FAZ proteins in the FAZ membrane and flagellum domains but had little effect on FAZ proteins in the cell body domain.
(A-E) Images of cells expressing FAZ proteins tagged with mChFP (magenta) representing the different FAZ domains in parental and FAZ2 null mutant cells. The flagellum membrane protein SMP1 is tagged with eGFP (green) and the DNA is stained with Hoechst 33342 (cyan). The inset shows an enlarged image of the FAZ protein localisation. Scale bar, 5 μm. F) Schematic of the FAZ domain organisation and anterior cell tip structure in the parental and FAZ2 null mutant cells.
Fig 5.
Flagellum-to-flagellum connections contained FAZ membrane domain proteins.
(A-E) Images of FAZ2 null mutant cells connected via their flagella expressing FAZ proteins tagged with mChFP (magenta) representing the different FAZ domains. The flagellum membrane protein SMP1 is tagged with eGFP (green) and the DNA is stained with Hoechst 33342 (cyan). The enlarged image shows the flagellum-to-flagellum connection in detail, with FAZ5 and FLA1BP clearly overlapping with the connection. Scale bar, 5 μm.
Fig 6.
Flagellum-to-flagellum connections were mediated by a small membrane bound bridge structure.
A, B) Conventional scanning electron micrographs of parental (A) and FAZ2 null mutant (B) cells through the cell cycle. The connection is formed between the flagella (white arrow) as soon as the new flagellum exits the cell body. Scale bar is 5 μm. C) High-resolution scanning electron micrographs showing the bridge that mediates flagellum-to-flagellum connection (the white box indicates the position of the area enlarged beneath). Scale bar, 5 μm (A, B, C image on the left) and 500 nm (C, lower panel). D) Slices through the tomogram showing the connections (white arrow) between the flagellum and the connecting bridge structure. The asterisks indicate the electron density within the connecting structure. Scale bar, 200 nm. E) Model of the connection generated from tomogram of connected flagella in the FAZ2 null mutant.
Fig 7.
FAZ2 deletion severely affected the ability of Leishmania to proliferate and develop in the sand fly and dramatically reduced pathogenicity in the mouse.
A) Analysis of sand fly infections using parental, FAZ2 null mutant and FAZ2 add-back cells. After 1–2 days post blood meal and 6–8 days post blood meal sand flies were dissected (number indicated above the column) and the parasite load in each fly was measured as heavy (1000+ parasites), moderate (100–1000 parasites), weak (1–100 parasites). This is the combined data from two independent sand fly infection experiments. B) Representative electron micrographs of longitudinal section through the flagellar pocket of a parental cell and FAZ2 null mutant axenic amastigotes. (i) represents flagellar pocket length, (ii) represents flagellum length, (iii) represents width of the flagellum at the constriction point. Scale bar is 1000 nm. C) Graphs for the 3 measurements in (B) with the mean plotted and error bars showing standard deviation for parental and FAZ2 null mutant axenic amastigotes. For flagellum length–parental cells n = 16, FAZ2 null mutant n = 24; for flagellar pocket length–parental cells n = 16, FAZ2 null mutant n = 27; for flagellum width–parental cells n = 18, FAZ2 null mutant n = 29. D) Measurement of mouse footpad lesion size during an 8-week infection time course with parental, FAZ2 null mutant and FAZ2 add-back cells. Error bars indicate standard deviation. A two-tailed unpaired t-test was used for the pairwise comparisons of the parental versus FAZ2 null mutant and FAZ2 null mutant versus FAZ2 add back. E) Measurement of parasite burden at the end of the 8-week infection time course in the footpad lesion and the lymph node for the parental, FAZ2 null mutant and FAZ2 add back cells. Parasite number from each infection is plotted with the mean and the 95% SEM interval indicated, and p values were calculated using a two-tailed unpaired Student’s t-test (n = 5).
Fig 8.
Model for the generation of the flagellum-to-flagellum connections.
The parental Leishmania promastigote flagellar pocket is shown from two different angles, giving a side-on and plan view of the FAZ. In the plan view of the FAZ, all the FAZ domains are aligned one on top of the other: flagellum domain on top, next the intermembrane domain, then finally the cell body domain. In the parental cell the new flagellum and FAZ (all domains) assemble alongside the existing flagellum/FAZ structure. In the FAZ2 null mutant the initial assembly of the new flagellum and new FAZ (all domains) is likely to occur alongside the remnant of the cell body FAZ domain of the old FAZ in the expected position. However, without FAZ2 the link between the cell body FAZ domain and the intermembrane and flagellum FAZ domains is not formed or is weak. Thus as the new flagellum and FAZ continue to assemble, the flagellum and intermembrane FAZ domains are ‘released’ from their link to the FAZ cell body domain and move along with the assembling new flagellum within an anterior cell tip extension. This anterior cell tip extension on the new flagellum is connected to an existing cell tip extension along the old flagellum. As the new flagellum continues to grow this structure extends farther until it eventually separates from the cell body to leave the two flagella connected by a membranous bridge structure.