Redistribution of FLAgellar Member 8 during the trypanosome life cycle: Consequences for cell fate prediction

Abstract

The single flagellum of African trypanosomes is essential in multiple aspects of the parasites' development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms of Trypanosoma brucei, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other parasite cycle stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian-infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating the remodelling of an existing flagellum. In the tsetse cardia, FLAM8 further localises to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum were seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered a meta-marker of the flagellum stage and maturation state in trypanosomes.

Keywords: FLAM8; Trypanosoma brucei; differentiation; division; elongation; flagellum; remodelling.

FIGURE 1

FLAM8 is differentially distributed along the entire flagellum in mammalian forms. (a) Immunofluorescence on methanol‐fixed AnTat EYFP::FLAM8 BSF in culture, using the anti‐GFP (green), the anti‐axoneme marker mAb25 (magenta), DAPI staining of DNA content (blue). Scale bar is 5 μm. (b) IFA on methanol‐fixed AnTat EYFP::FLAM8 BSF showing that at the base of the flagellum FLAM8 (anti‐GFP signal in green) do not co‐localise with the transition zone component FTZC (in yellow) and initiates before the starting of the PFR (in magenta). White arrowheads indicate the beginning of the PFR signal. Scale bar is 5 μm. (c) FLAM8 is not localised to the flagellum membrane. IFA on methanol‐fixed AnTat EYFP::FLAM8 BSF (left panels), paraformaldehyde‐fixed (middle panels) and cytoskeletons (right panels) using the anti‐GFP antibody (green) and an antiserum against the arginine kinase of T. cruzi (magenta), DAPI staining of DNA content (blue). Arginine kinase (AK) signal, which is localised to the flagellum membrane, was almost completely lost after detergent extraction (right panels). Scale bar is 5 μm. (d) Structured illumination microscopy images (SIM) of FLAM8 (anti‐GFP antibody in white) with flagellar components, paraflagellar rod (PFR, magenta) and axoneme (green) showed in pleomorphic EYFP::FLAM8 PCF and BSF parasites (upper and lower panels, respectively). DAPI staining of DNA content appears in blue. Scale bars represent 1 μm. Images: white arrows show the old (OF) and the new flagellum (NF). Images are representative of three independent experiments for panels (a), (b) and (c), and two independent experiments for panel (d)

FIGURE 2

FLAM8 is also a marker of the flagellum maturation in mammalian infective forms. (a) Immunofluorescence on AnTat EYFP::FLAM8 BSF in culture expressing FLAM8 fused to the EYFP protein in the N‐terminus. The axoneme (mAb25 antibody in magenta) shows the full length of the flagellum; FLAM8 was labelled using the anti‐GFP antibody (green), and DAPI staining of DNA content appears in blue. (b) Dot plot showing the quantification of FLAM8 intensity present at the proximal region in 1K1N, 1K1N with two flagella (1K1N 2FL), 2K1N and 2K2N BSF parasites, compared with the total length of the flagellum measured by the use of the axoneme marker mAb25. The number of cells used for the quantification (N) is indicated above the graph. (c) Linear correlation between the FLAM8 signal and the length of the axoneme in 1K1N, 1K1N 2FL, 2K1N and 2K2N trypanosomes. (d) IFA on methanol‐fixed AnTat EYFP::FLAM8 parasites left untreated (top panels) or treated for 6 hr with teniposide (bottom panels) stained with anti‐GFP (green), mAb25 antibody (magenta) and DAPI labelling DNA (blue). (e) Ratio between the FLAM8 fluorescent signal at the tip in the new and old flagellum in cells treated or non treated with teniposide for 6 hr. Statistically significant differences upon linear regression analysis followed by unpaired t‐test are indicated with two stars (p < .005), and the number of cells used for quantification is indicated below the graph. Images: white arrowheads show the old (OF) and the new flagellum (NF). Scale bar is 5 μm. Results are given as means (±standard deviation, SD) of biometric measurements of cells from two independent experiments

FIGURE 3

FLAM8 concentration to the tip reflects the remodelling of an existing flagellum during BSF to PCF differentiation. (a) Representative images of AnTat 1.1E SL and ST parasites isolated from infected BALB/c mice and undergoing ex vivo differentiation from non‐dividing stumpy forms (left panels) into early procyclic trypanosomes at 27°C (panels named 2, 4, 12 and 24 hr). Immunofluorescence images of methanol‐fixed parasites stained with anti‐FLAM8 antibody (green), anti‐axonemal mAb25 (magenta), and antibodies against specific surface markers: anti‐PAD1 to stain ST forms (in cyan), and anti‐procyclin labelling early PCF parasites (in pink). DAPI staining appears in blue. Scale bar is 5 μm. (b) Quantification of the FLAM8 intensity at the tip of the flagellum during ex vivo differentiation into insect procyclic forms. Statistically significant differences after one‐way ANOVA followed by Tukey–Kramer post‐test are indicated with three (p < .001) and four stars (p < .0001). The number of parasites used for quantification (N) is indicated below the graph. Results are given as means (±standard deviation, SD) of biometric measurements of cells from one experimental infection and two biological replicates. AU, arbitrary units

FIGURE 4

FLAM8 accumulates differently in dividing procyclics from posterior versus anterior midgut. Glossina m. morsitans tsetse flies were fed with a blood meal containing pleomorphic AnTat 1.1E ST parasites. The flies were dissected at distinct time points in order to isolate different MG regions and look for PCF trypomastigote stages. (a–c) Infected tsetse flies were dissected at day 3 for PMG region isolation. (a) Representative immunofluorescence images of methanol‐fixed 2K1N (upper panels) and 2K2N (bottom panels) dividing parasites stained with anti‐FLAM8 antibody (green), anti‐axoneme marker mAb25 (magenta) and DAPI staining (blue). White arrowheads indicate the old and new flagella (OF and NF, respectively). Scale bar is 5 μm. (b) Quantification of FLAM8 intensity at the flagellum tip of 1K1N parasites and both OF and NF of dividing 2K1N and 2K2N trypanosomes. Statistically significant differences are indicated with three (p < .001) and four stars (p < .0001). (c) Ratio between FLAM8 intensity in the new flagellum (NF) and the old flagellum (OF) in 2K1N and 2K2N trypanosomes. Statistically significant differences are indicated with four stars (p < .0001). Black dotted line indicates signal at 100% ratio. (d–f) Tsetse flies receiving an infective blood meal were dissected after 11 days for anterior midgut (AMG) isolation. (d) Representative immunofluorescence images of methanol‐fixed 2K1N (upper panels) and 2K2N (bottom panels) procyclic cells showing anti‐FLAM8 (green), mAb25 signals (magenta) and DAPI staining (blue). White arrowheads indicate the old flagellum (OF) and the new flagellum (NF) of dividing trypanosomes. Scale bar is 5 μm. (e) Dot plot showing FLAM8 signal quantification at the flagellum tip of 1K1N parasites and 2K1N and 2K2N procyclic cells at both mature (OF) and growing flagella (NF). Statistically significant differences are indicated with two (p < .01) and four stars (p < .0001). (f) Ratio between FLAM8 intensity in the new flagellum (NF) and the old flagellum (OF) in 2K1N and 2K2N trypanosomes. Statistically significant differences are indicated with four stars (p < .0001). Black dotted line indicates signal at 100% ratio. In (b) and (e), the number of parasites used for quantification (N) is indicated below the graphs. Statistical analyses include one‐way ANOVA followed Tukey–Kramer post‐hoc tests for panels (b) and (e), and unpaired t‐tests for panels (c) and (f). Results are given as means (±standard deviation, SD) of biometric measurements of cells from three independent experiments and at least three biological replicates. n.s., not significant

FIGURE 5

FLAM8 distribution at the flagellum tip extends with the elongation of the flagellum in long trypomastigote parasites in the midgut. Tsetse flies were fed with freshly‐differentiated AnTat 1.1E stumpy parasites and after 14 days the entire digestive tract including the cardia/proventriculus was isolated for immunofluorescence analyses and further quantification. (a) Selected images of methanol‐fixed procyclics, long PCF, LT and LT from the cardia stained with mAb25 axonemal marker (magenta), anti‐FLAM8 antibody (green) and DAPI for DNA content (blue). Upper line: midgut stages (green) versus trypomastigotes found in the cardia (red). (b) Dot plot showing the FLAM8 intensity at the tip of procyclic parasites, long PCF and long trypomastigotes (LT) recovered from the midgut and the cardia. Statistically significant differences upon one‐way ANOVA followed by Tukey–Kramer post‐test are indicated with four stars (p < .0001). The number of parasites used for quantification (N) is indicated below the graph. (c) The mAb25 axonemal signal was used for flagellum length quantification and further correlation with the FLAM8 amount at the tip of specific parasite stages. Results are given as means (±standard deviation, SD) of biometric measurements of cells from two independent experiments and at least three biological replicates

FIGURE 6

FLAM8 is redistributed along the trypanosome flagellum twice during the parasite cycle in the insect vector. (a) AnTat 1.1E stumpy parasites were used to infect Glossina m. morsitans tsetse flies in order to collect fly tissues (midgut, cardia and salivary glands), and look for all developmental stages after 30 days of infection. Immunofluorescence images using anti‐FLAM8 antibody (green), anti‐axonemal marker mAb25 (magenta) and DAPI staining (blue). White dashed‐line boxes show a two‐fold magnification of the distal tip in all parasite stages. Scale bar is 5 μm. (b) and (c) The ratio of intensity of FLAM8 fluorescence at the distal tip (b) and along the entire flagellum (c) related to flagellum length was quantified in all trypanosome morphotypes found within the midgut, cardia and the salivary glands of infected tsetse flies. The number of cells used for the quantification (N) is indicated below the graphs. Statistically significant differences after one‐way ANOVA followed by Tukey–Kramer post‐tests are indicated with two (p < .01) and four stars (p < .0001). Results are given as means (±standard deviation, SD) of biometric measurements of cells from three independent experiments and at least three biological replicates. AE, attached epimastigote; DE, dividing epimastigote; LE, long epimastigote; LT, long trypomastigote; MT, metacyclic; PCF, procyclic; SE, short epimastigote

FIGURE 7

FLAM8 is differentially distributed during the asymmetric division in the salivary glands. (a) Representative images of ‘Epi‐Trypo’ transition in the salivary glands. Methanol‐fixed AnTat 1.1E parasites were stained with anti‐FLAM8 antibody (green), anti‐axonemal marker mAb25 (magenta) and DAPI staining (blue). White arrowheads within the ‘Epi‐Trypo’ panel indicate old (OF) and new (NF) flagellum. Scale bar is 5 μm. (b) Quantification of FLAM8 fluorescence intensity along the flagellum related to the length of the axoneme. The number of parasites considered for quantification (N) is indicated below the graphs. Statistically significant differences after one‐way ANOVA followed by Tukey–Kramer post‐test are indicated with two (p < .01) and four stars (p < .0001). Results are given as means (±standard deviation, SD) of biometric measurements of cells from four independent experiments and at least three biological replicates. 2K1N, ‘Epi‐Trypo’ parasite during the asymmetric division; AE, attached epimastigote; MT, metacyclic; preMT, pre‐metacyclic

FIGURE 8

FLAM8 is a marker of the flagellum maturation state that predicts the fate of a trypanosome during the parasite cycle. In the mammalian host, proliferative slender (SL) parasites divide in order to colonise the intravascular and extravascular compartments. This symmetric division gives rise to one daughter cell that will undergo another round of division or that will stop proliferation and differentiate into arrested stumpy trypanosomes (ST). Both SL and ST trypomastigotes exhibit FLAM8 along the full flagellum length. During a blood meal on an infected host, a tsetse fly will ingest a pool of SL and ST cells, but only the latter will be capable of surviving in the new host environment. In the PMG, ST will rapidly transform into proliferative procyclic (PCF) trypomastigotes. This event will be characterised by the dramatic remodelling of FLAM8 distribution in the existing flagellum, since PCF cells display a FLAM8 concentrated only at the very distal tip of the flagellum. In addition, 2K2N PCF cells dividing in the PMG express a lower amount of FLAM8 in the growing flagella, associated with a post‐division maturation of the NF in the daughter cell according to the grow‐and‐lock model. The daughter cell would then probably re‐enter another proliferation cycle in order to maintain the proliferative PCF population in the PMG. In contrast, dividing procyclic cells located in the anterior midgut present a higher amount of FLAM8 in the NF before cytokinesis, suggesting that the daughter cell would proceed to the next developmental step and differentiate into non‐proliferative long trypomastigotes (LT) by a drastic elongation of their flagella. These elongated trypomastigote parasites will subsequently enter the cardia in order to initiate a differentiation into epimastigote stages. During the asymmetric division performed by these long dividing epimastigotes (DE), two daughter cells are generated: a long epimastigote (LE), which is believed to die shortly after migration and/or cytokinesis and a short epimastigote (SE) that will further develop in the salivary glands. Unlike DE and LE, SE present FLAM8 along the entire length of the flagellum, evidencing a second redistribution of the protein in vivo. Once in the salivary glands, SE will differentiate into stages that will attach to the salivary epithelium, keeping FLAM8 along their entire flagellum. Interestingly, during the asymmetric division carried out by ‘Epi‐Trypo’ dividing AE, the daughter cell inheriting the NF exhibits a higher amount of FLAM8 as compared to the OF. This daughter cell will then differentiate into pre‐metacyclic (preMT) parasites and, finally, into infective metacyclic trypomastigotes, with FLAM8 being present along the entire flagellum length. Red dotted boxes show the magnification of particular trypanosome areas indicated with numbers. Colour‐code: the axoneme is depicted in black while the FLAM8 distribution is shown in green in the OF and in yellow in the NF. FLAM8_NF, FLAM8 amount in the new flagellum; FLAM8_OF, FLAM8 amount in the old flagellum; K, kinetoplast; N, nucleus