The ALBA protein family in T. brucei
The genomes of T. brucei, Trypanosoma cruzi, and Trypanosoma vivax encode four proteins containing an ALBA domain, whereas only two are found in Trypanosoma congolense and in all Leishmania species. In T. brucei, two ALBA genes are found on chromosome 4: ALBA3 (Tb927.4.2040) and ALBA4 (Tb927.4.2030) and two on chromosome 11: ALBA1 (Tb11.02.2040) and ALBA2 (Tb11.02.2030; Supplemental Figure S1). ALBA1 and ALBA2 are small proteins of 12 and 14 kDa that contain only the ALBA domain (Pfam PF01918) and that show 53% identity on the protein level between each other. ALBA3 and ALBA4 are very divergent from ALBA1 and ALBA2, with which they share only 16% overall identity, restricted to the ALBA domain. ALBA3 and ALBA4 show high conservation between them, with 85% identity at the DNA level (Supplemental Figure S2A). The encoded proteins have a molecular weight of 21 and 25 kDa, respectively and contain, in addition to the ALBA domain, a C-terminal stretch of multiple RGG repeats that are believed to be important in nucleotide binding. This study focuses on the investigation of ALBA3 and ALBA4 since they are more likely to be true orthologues of ALBA proteins found in metazoa (Supplemental Figure S1B).
ALBA3/4 are cytosolic proteins that aggregate in mRNA-containing granules upon starvation
To investigate ALBA3 and ALBA4, both full-length proteins were expressed as glutathione S
-transferase (GST) fusions and used to produce antisera in mice. Western blot analysis was carried out on total extracts of cultured procyclic trypanosomes of strain Lister 427 and confirmed that both proteins were expressed. They migrated at positions corresponding to their predicted size of 21 and 25 kDa for ALBA3 and ALBA4, respectively (). Of the 16 antisera produced, most of them recognized both bands with various efficiencies (see, e.g., ), whereas one turned out to be more specific for ALBA3 () and another one for ALBA4 (). Comparable levels of ALBA3 and ALBA4 proteins were found in the bloodstream form in culture (). Next, cell lines expressing yellow fluorescent (YFP) fusion proteins with either ALBA3 or ALBA4 were generated in procyclic cells of Lister 427 and AnTat1.1 strains (this latter strain is fully competent to infect tsetse flies; see later discussion; Le Ray et al., 1977
). The plasmids were linearized within the ALBA3
coding sequence to target integration and subsequent expression of the fusion construct from the endogenous locus (). Protein expression was verified by Western blot using either the anti-ALBA3–specific antibody () or the anti-ALBA4–specific antibody (). The YFP-tagged versions ran at positions in agreement with their molecular weights of 46 kDa (ALBA3::YFP; ) and 50 kDa (ALBA4::YFP; ), respectively. These results confirmed the specificity of the ALBA3 and ALBA4 antibodies. Moreover, as expected from the endogenous tagging procedure, the amount of untagged protein seemed reduced for both ALBA3 and ALBA4 ().
FIGURE 1: ALBA protein expression and localization in wild-type and ALBA::YFP–expressing cell lines. (A–C) Reactivity of different ALBA antibodies assessed by Western blot in the indicated cell lines (2 μg of total protein extracts per lane). (more ...)
Ten anti-ALBA antibodies were then used by immunofluorescence analysis (IFA) to evaluate the localization of ALBA3/4 proteins. After paraformaldehyde (PFA) fixation, all antibodies tested produced a clear signal evenly distributed in the cytoplasm of the cultured procyclic form (). ALBA3/4 were absent from the nucleus and the kinetoplast at all steps of the cell cycle. In contrast, signal was lost upon methanol fixation, suggesting that ALBA3/4 are cytosolic proteins that are solubilized upon membrane removal. Finally, observation of the ALBA::YFP direct fluorescence in live cells confirmed the exclusively cytoplasmic location and the absence of visible changes in the staining pattern during the cell cycle ().
ALBA3 and ALBA4 are therefore unlikely to be involved in DNA binding, but as they possess RGG repeats, they could interact with RNA. In trypanosomes, several RNA-interacting proteins accumulate into cytoplasmic foci that harbor mRNA and are formed in starvation conditions or upon heat shock (Cassola et al., 2007
; Kramer et al., 2008
). The localization of ALBA3/4 was investigated in these two stress conditions, first by IFA with the anti-ALBA3 and an antibody against DHH1, a recognized marker of these granules (Kramer et al., 2008
). In contrast to DHH1, the ALBA signal did not show discrete foci upon heat shock (). However, the ALBA signal was clearly concentrated in foci after starvation stress, where it colocalized with DHH1 ().
FIGURE 2: Localization of ALBA proteins in stress conditions and colocalization studies. Wild-type procyclic trypanosomes of the Lister 427 strain were subjected to different stresses: either (A) heat shock (42°C for 2 h) or (B) starvation (incubation in (more ...)
Because the access of the antibody to the antigen in the foci could be limited, these results were confirmed by analyzing the cell lines expressing endogenous ALBA3::YFP and ALBA4::YFP fusion proteins. In starvation conditions, both YFP fusion proteins started to aggregate into cytoplasmic foci as soon as after 10 min and reached a maximum accumulation after 2 h (unpublished data), confirming the IFA results. To further define ALBA localization, a mCherry::DHH1 construct (Kelly et al., 2007
) was added to these cell lines, revealing that both proteins colocalized in cytoplasmic granules after 2 h of nutritional stress ().
The colocalization of ALBA with a described RNA-binding protein in cytoplasmic foci prompted the investigation of poly(A+) RNA, which was shown to colocalize with the DHH1 protein upon starvation stress (Cassola et al., 2007
). Therefore, the RNA–fluorescence in situ hybridization (FISH) method to detect poly(A+) RNA was coupled to IFA using the anti-ALBA3 antibody (). Whereas conventional epifluorescence microscopy suggested perfect colocalization, the analysis of Z-stacks by confocal microscopy revealed three types of foci in starved parasites: 1) those containing poly(A+) RNA and ALBA protein displaying equal signal intensity, 2) those containing poly(A+) RNA but no detectable ALBA protein, and 3) those showing a signal for ALBA protein but no detectable poly(A+) RNA signal. In conclusion, both ALBA proteins formed foci upon starvation stress conditions, colocalized with the DHH1 protein, and showed partial colocalization with poly(A+) RNA.
Depletion of ALBA3/4 blocks cell growth and induces morphological modifications
To evaluate the importance of ALBA3 and ALBA4 for trypanosomes, their expression was depleted either simultaneously or individually using RNAi in 29-13 procyclic parasites in culture. The first 400 base pairs of the ALBA3 and ALBA4 coding region show 94% identity (Supplemental Figure S2A) and were targeted for simultaneous knockdown of ALBA3 and ALBA4. The alignment of all ALBA DNA sequences (including the region of highest conservation; Supplemental Figure S2B) shows that the nucleotide identity is so low (maximum stretch of identity is limited to four consecutive nucleotides) that the possibility of cross-RNAi against ALBA1 or ALBA2 can be formally ruled out. The silencing efficiency of ALBA3/4 was assessed by semiquantitative reverse transcription-PCR (RT-PCR) () and by Western blot using the antibody recognizing both ALBA3 and ALBA4 () or an antibody recognizing exclusively ALBA3 (). The mRNA levels of both ALBA3 and ALBA4 were significantly reduced after 2 d of induction (). ALBA protein amounts dropped significantly starting from day 2 () and remained low after up to 7 d of continuous induction of RNAi (unpublished data). The anti-ALBA3 antibody was used in IFA to show protein down-regulation at the single-cell level (Supplemental Figure S3, B–D). The number of ALBA-positive cells decreased rapidly until day 3, when almost 100% of the cells were ALBA negative (Supplemental Figure S3B). Once it was proven that the ALBA proteins were efficiently knocked down in the ALBA3/4RNAi cell line, the growth in culture was monitored with and without tetracycline (). After a comparable proliferation in the first 2 d, the induced cells showed slightly slowed down growth after the first dilution step, but almost ceased to duplicate after the second dilution step, in contrast to the noninduced control, which subsequently needed dilution every 2 d ().
FIGURE 3: ALBA3/4RNAi cells exhibit defects in cell growth and morphogenesis. (A) Semiquantitative RT-PCR on total RNA of the control 427 strain or the ALBA3/4RNAi cell line induced for 2 d, using specific primers for ALBA3 or ALBA4. (B, C) Western blot with 2 (more ...)
Trypanosomes in culture divide by binary fission, and the cell cycle phases are recognized by number and position of DNA-containing organelles: G1/S (1 kinetoplast [K] and 1 nucleus [N]), G2/M (2K1N), and postmitotic cells (2K2N) (). Aberrant cell types were also recorded: 1K2N parasites, cells without nucleus called zoids (1K0N; Robinson et al., 1995
), and cells with multiple nuclei (>2N). The noninduced state was considered as the control situation in which the population was split into 79% 1K1N, 14% 2K1N, and 4% 2K2N cells and a minority of various aberrant cell types (<3%). No major changes in these proportions were observed up to 3 d of induction, in agreement with the growth curve. However, after the fourth day of RNAi induction, the proportion of 2K1N cells decreased, whereas the percentage of zoids went up, reaching 15% after 6 d, indicating cell cycle defects (Ploubidou et al., 1999
). In contrast to many RNAi mutants in T. brucei
, we did not observe the emergence of multinucleated cells ().
To further investigate the consequences of the ALBA3/4 knockdown, cells induced at various time points were fixed and stained by IFA with the anti-ALBA3 antibody. The cells devoid of ALBA staining exhibited two main, striking phenotypes (). First, they showed increased length up to 40 μm (noninduced cell length in the 1K1N state is ~25 μm) (, star). As early as 2 d after the induction of protein knockdown, 24% of the cells showed an elongated posterior end, reaching up to 50% after 5 d (, graph). This phenotype was previously reported in some mutants and termed “nozzle” (Hendriks et al., 2001
; Li et al., 2003
; Hammarton et al., 2004
). However, a second phenotype specific to ALBA3/4RNAi
was observed: the nucleus frequently appeared in a posterior position relative to the kinetoplast (, arrowhead). The percentage of such cells increased constantly during the course of RNAi induction (, histogram). ALBA3/4RNAi
cells induced for 4 d were analyzed by scanning electron microscopy after extraction of the membrane, allowing direct visualization of the microtubule cytoskeleton corset and revealing the elongated posterior end with apparently correctly assembled microtubules (Supplemental Figure S3, E and F). In addition, we were able to have an inside view of a nucleus more posterior to the two kinetoplasts, combining the two phenotypes observed by light microscopy (Supplemental Figure S3F).
-induced cells were stained with the YL1/2 antibody, which recognizes newly assembled tyrosinated α-tubulin (Kilmartin et al., 1982
; Sherwin and Gull, 1989
; ), to evaluate whether the elongation in nozzle cells was due to an active polymerization of microtubules at the posterior pole. The staining was found at the basal bodies and the daughter flagellum, as well as at the posterior end in both control and ALBA3/4RNAi
-induced cells, but ALBA3/4RNAi
cells with an elongated posterior end displayed by far the brightest signal (, stars). In T. brucei
, the length of the single flagellum that extends along the cell body controls cell size (Kohl et al., 2003
). It was therefore measured in the ALBA3/4RNAi
cells to exclude a contribution to cell elongation. Regardless of the cell length and the RNAi induction time point (days 4–6), the length of the flagellum remained in the normal range (unpublished data). In contrast, the distance from the kinetoplast to the posterior end correlated in a linear relationship with cell length, which can be linked to the exhaustive microtubule elongation at the posterior pole (unpublished data).
To assess whether these phenotypes were the result of silencing of ALBA3 or ALBA4 alone or of the combination of ALBA3 and ALBA4, constructs allowing expression of double-stranded (ds) RNA targeting specifically ALBA3 or ALBA4 (Supplemental Figure S3A) were produced and transfected in trypanosomes. Potent silencing of ALBA4 was achieved, but this did not result in a visible phenotype (unpublished data). In contrast, generation of a cell line silencing ALBA3 was very difficult either because of significant expression of ALBA3
dsRNA even before the addition of tetracycline or because of the emergence of cells that did not silence ALBA3 but proliferated rapidly (unpublished data). This was reported previously for several RNAi experiments (Chen et al., 2003
) and prevented full investigation of the cell line. These data suggest that ALBA3 could be essential for procyclic trypanosomes. In summary, procyclic cells depleted of ALBA3/4 proteins elongated by active polymerization of microtubules at their posterior end relocated their nucleus to the posterior side of the kinetoplast and became arrested in the cell cycle.
ALBA3/4 expression profile during trypanosome development in the tsetse fly
All the foregoing observations suggest that ALBA3/4 knockdown mimics several typical changes happening in cells that undergo a trypomastigote-to-epimastigote differentiation during parasite development in the anterior midgut of a tsetse fly. However, this differentiation step is not reproducible in the laboratory. To assess ALBA protein levels in vivo during the parasite cycle, tsetse flies were fed with procyclic parasites of the AnTat 1.1 strain. Flies were dissected at various time points of infection, and the obtained parasites were fixed in PFA to perform IFA with the selected anti-ALBA3 antibody. The procyclic form (PC) obtained from the fly midgut showed a cytoplasmic ALBA staining () equivalent to that observed with the cultured form (). As the cell elongated to differentiate into the nonproliferative mesocyclic trypomastigote form (MS), no visible changes in ALBA signal intensity or profile were detected (). The following stage in the parasite cycle, the mesocyclic to epimastigote form (MS-E), is found mainly in the proventriculus of the fly and does not proliferate but is characterized by an elongated nucleus migrating toward the posterior end of the cell (Vickerman et al., 1988
; Sharma et al., 2008
; Rotureau et al., 2011
). The MS-E parasites showed strongly reduced ALBA level (, and Supplemental Figure S4). The ALBA level remained very low in cells that had adopted the epimastigote configuration, including after kinetoplast duplication and mitosis in the asymmetrically dividing epimastigote form (DE) (). After division, the short form (SE) reacquired ALBA signal, whereas the long one (LE) remained negative (). Finally, all the forms in the salivary glands were positive for ALBA: the salivary gland epimastigotes (SGEs) and the infective metacyclic trypomastigote parasites (MTs) ().
FIGURE 4: ALBA3/4 expression level during parasite development in the tsetse fly. (A–J) Evolution of ALBA3/4 expression level assessed by IFA with the anti-ALBA3 antibody (left) and counterstained with DAPI (blue in the phase contrast image). ALBA signal (more ...)
These data indicate a precise control of ALBA3 and ALBA4 during trypanosome development in the fly. However, quantification of the results is challenging since these stages are present in low numbers and difficult to access, and, moreover, the necessary use of PFA fixation means that even fewer parasites are available for analysis (cells fixed in these conditions do not adhere well on microscope slides). We therefore chose to work with live cells expressing YFP fusion proteins, which also avoids limitations resulting from fixation approaches. Hence, tsetse flies were fed with either one of the ALBA::YFP AnTat1.1 cell lines and dissected at different time points of infection. For both strains, the rates of infection in the midgut (MG) and salivary glands (SG) were comparable with those for the parental wild-type AnTat1.1 strain (control MG, 40%, SG 10.7%; ALBA3::YFP MG, 54%, SG 10.3%; ALBA4::YFP, MG 33%, SG 11%). shows a typical example of ALBA3::YFP cells released from the proventriculus: although mesocyclic parasites showed strong ALBA signal, the mesocyclic to epimastigote forms seemed almost negative. In this experiment, parasites were released from fly tissues into phosphate-buffered saline (PBS), demonstrating that the formation of cytoplasmic starvation granules also occurs in parasites issued from an infection ().
Because endogenous ALBA fusion protein expression reflected the results obtained by IFA, ALBA expression profile was monitored by live videomicroscopy. However, parasites can only be seen upon dissection and are short lived after fly sacrifice, restricting time available for analysis. We selected to acquire images by analogue video recording. This is not a digital system, and absolute quantification cannot be performed, but three categories of cells can be clearly defined: negative (−), positive (+), and bright (++). By this mean, it was possible to analyze a sufficiently large number of cells (1216 for ALBA3::YFP and 775 for ALBA4::YFP) not reachable otherwise. The differentiation stage was determined by phase contrast imaging (for morphology) coupled with DNA staining by 4′,6-diamidino-2-phenylindole (DAPI), a dye that penetrates easily in live trypanosomes (). Supplemental Movie S1 shows representative cells for each ALBA3::YFP parasite stage found in the fly, and Supplemental Movie S2 shows fields of cells of the same strain to illustrate how relative fluorescence intensity was evaluated.
All ALBA3::YFP cells showed a bright fluorescent signal at the PC stage, whereas the MS parasites were more heterogeneous (40% bright and 60% positive green cells; ). The ALBA3 fluorescence intensity dropped further during the transition to the epimastigote form, with almost 80% of the parasites being negative. This was even more pronounced in the DEs (>90% of ALBA3::YFP negative cells). The ALBA signal was recovered in the SEs, whereas LEs remained negative, confirming the IFA data (). Finally, all trypanosome forms found in the salivary glands were strongly positive for ALBA3 ().
The expression profile of ALBA4::YFP turned out to be very similar to that of ALBA3 ( and Supplemental Movie S3), with the exception of the DE, with 35% of positive fluorescent cells in ALBA4 versus 10% in ALBA3. This could be explained by a faster reemergence of ALBA4 protein in the future SE before cytokinesis is completed, going along with the higher percentage of SE-positive cells for ALBA4 versus ALBA3 ().
Taken together, the combination of IFA and live videomicroscopy analysis showed a defined expression profile of ALBA3 and ALBA4 proteins during the parasite cycle, marked by a significant drop at the trypomastigote to epimastigote transition.
Constitutive overexpression of ALBA3 proteins impairs differentiation
To understand the significance of the down-regulation of ALBA3/4 in the proventricular stages, we sought to overexpress them. However, regulatory elements required for expression at these particular stages are unknown. Hence, we selected to use the pHD67E vector, which had been reported to confer GFP reporter expression throughout the parasite cycle (Bingle et al., 2001
). This construct was targeted to the rDNA locus, and the reporter GFP protein was expressed under the control of the EP procyclin promoter. Either ALBA3
coding region was fused upstream of the GFP
gene into this vector. The expression as GFP fusion was necessary to control the level of overexpression in individual cells during parasite development in the fly. Procyclic trypanosomes of the AnTat1.1 strain were transfected with the plasmid pHD67E as control or with pHD67EALBA3 and pHD67EALBA4. Protein expression levels were first analyzed by Western blot in cultured procyclic cells using the specific anti-ALBA3 or anti-ALBA4 antibodies (). Fusion proteins exhibited the expected electrophoretic mobility, and their abundance appeared to be equivalent to that of the endogenous ALBA3 or ALBA4, meaning that these cells were expressing double the amount of ALBA3 or ALBA4 (). This exogenous expression of ALBA::GFP did not lead to a down-regulation of the endogenous ALBA protein, but additional protein bands of intermediate size were detected with both the anti-ALBA3 and anti-ALBA4 antibodies (), possibly corresponding to degradation products of the fusion protein, a feature not seen during the endogenous tagging experiments (). To compare the expression levels between cell lines, protein samples were probed with an anti-GFP antibody (). ALBA4::GFP was expressed to a level comparable to that of the control GFP, whereas ALBA3::GFP turned out to be less abundant, despite the use of the same expression system. In the GFP control cells, direct observation of fluorescence in live or fixed cells () revealed that GFP expression was heterogeneous from one cell to the other and that the signal was detected in the cytoplasm as well as in the flagellum and the nucleus (, top). In contrast, ALBA3::GFP (unpublished data) and ALBA4::GFP fusion proteins (, bottom) were found exclusively in the cytoplasm. All three cell lines grew normally in culture, and cells did not exhibit any particular phenotype.
FIGURE 5: Overexpression of ALBA3::GFP or ALBA4::GFP and consequences for fly infection. (A) Western blots with 2 μg of total protein samples of AnTat1.1 WT cells carrying a GFP gene (control GFP) or an extra copy of either ALBA3::GFP or ALBA4::GFP. Blots (more ...)
Several groups of flies were infected with each of the strains, and dissection was undertaken at late time points (days 25–30), when control infections should have reached maturity in the salivary glands. Infection rates were obtained by grouping data of nine independent experiments (). For the GFP control strain, 52% of the flies showed midgut infections, and salivary gland infections were detected in 12% of the flies (), which correspond to the levels observed in our laboratory for the AnTat1.1 wild-type strain (Rotureau et al., 2011
). One-third of the established infections in the midgut led to a mature salivary gland infection. The ALBA3::GFP strain turned out to be as efficient as the control in developing midgut infections (53%). The level of salivary gland infections was slightly lower (6%; meaning that only 10% of the established infections in the midgut led to a mature infection in the fly saliva). It should be noted that these flies showed an unusually high number of midgut parasites in comparison to other midgut infections observed for the same cell line. For the ALBA4::GFP strain, 50% of the flies were infected in the midgut, and 17% showed parasites in the salivary glands, values that are very close to the control situation, with one-third of midgut infections leading to a mature development.
Live trypanosomes from all three strains were investigated by videomicroscopy as described earlier (Supplemental Movie S4). Starting with a high proportion of green fluorescent procyclic parasites (78–98%), the green signal evolved differently during progression in the parasite cycle (). In the GFP control cell line, 80% of the cells were strongly positive at the midgut PC or MS stages, and negative parasites were rare (). The general trend in all proventricular stages was a drop in the proportion of strongly positive parasites, but the abundance of GFP-negative parasites remained constantly low, ~20%. Cells in the salivary gland were positive in most cases (SGE and MT). In summary, the reporter GFP protein was present in 80 to nearly 100% of the cells in all stages of the parasite cycle, although its abundance was variable (). In contrast to the endogenous tagging experiments, different results were obtained for cells overexpressing ALBA3::GFP () or ALBA4::GFP (). Infections with the ALBA4::GFP strain were characterized by a majority of cells expressing a high level of the fusion protein, whereas negative parasites were a minority in all stages investigated, with the exception of long epimastigotes (). This profile is similar to the expression of the control GFP alone. In contrast, proventricular parasites resulting from ALBA3::GFP infections showed a higher proportion of negative cells: 35% for the MS-E and up to 47% for the DE, whereas the percentage of positive SE was comparable to the results obtained for ALBA4::GFP. Only a few cells could be analyzed in the salivary glands due to reduced parasite load, but all turned out to be negative or weakly positive (), in contrast to the other two cell lines (). These results show that overexpression of the ALBA4 protein as GFP fusion is maintained in each stage during progression of the parasite cycle without impairing the rate of fly infectivity. In contrast, although the same expression system was used, the ALBA3::GFP protein was down-regulated at the transition from trypomastigote to epimastigote stage, like the endogenous ALBA3 protein. Moreover, parasites expressing large amounts of ALBA3::GFP proteins were not observed in the salivary glands, suggesting that strong overexpressers cannot complete the final part of the parasite cycle.
During the live video analysis of the ALBA3::GFP cells (but not of the GFP control or in the ALBA4::GFP strain) in the anterior midgut and proventriculus, our attention was attracted by a significant proportion of parasites that showed atypical cell diameter and DNA organelle positioning (). Several morphometric parameters were measured: the length of the parasite (from the posterior end of the cell to the flagellum tip), the cell diameter, the distance between kinetoplast and nucleus centers, and the length of the nucleus. To avoid artifacts potentially caused by fixation methods, this analysis was performed on cells from the live movies of the GFP control and the ALBA3::GFP parasites (). Two populations were identified in the GFP control: MS cells with a fairly large diameter (1.2 μm) and with a round nucleus positioned close to the center of the cell (102 of 155 analyzed cells; ) and MS-E cells with a thinner diameter (0.9 μm) and an elongated, oval nucleus undergoing migration toward the posterior end of the cell (, left). These two phenomena were always found to occur in parallel but never uncoupled. Although typical MS (126 of 185 analyzed cells) and MS-E (18 of 185) were present, an atypical cell type (ATYP) was also observed in the ALBA3::GFP cell line (41 of 185). These cells presented a pronounced thin cell diameter (0.83 ± 0.06 μm) typical for MS-E but, strikingly, despite having the cell diameter of MS-E, they exhibited a nonelongated nucleus (3.18 ± 0.4 vs. 4.58 ±0.58 μm in MS-E) whose position was more anterior than that observed in MS-E. The distance to the kinetoplast was 6.84 ± 1.40 μm instead of 5.0 ± 1.14 μm ().
FIGURE 6: Characterization of the transition from mesocyclic trypomastigote to epimastigote stage in the GFP control and the ALBA3::GFP–expressing strain. Still images extracted from movies of cells of the AnTat1.1 strain expressing GFP control (A) or the (more ...)
In conclusion, this atypical form encountered in the ALBA3::GFP overexpressing strain displayed the morphology of an MS-E regarding the cell diameter, but the nucleus length and its distance to the kinetoplast were found to be comparable to the mesocyclic form. This suggests that the presence of the ALBA3::GFP protein delayed or inhibited migration of the nucleus toward the posterior end of the cell during the trypomastigote to epimastigote transition, further strengthening the hypothesis that ALBA3 is implicated in this differentiation process.