Fig 1.
Evaluation of different encystation protocols and ESV formation.
(A-C) Flow cytometry analysis of three different encystation protocols in regard to mature water resistant 16N cysts. (A) and (B) histograms represents the cellular distributions in ploidy generated with the standard two-step method and lipid starvation method respectively. The Uppsala encystation protocol (C) generated a higher proportion and yield of mature 16N cysts. Encystation kinetics in the newly developed protocol was evaluated by counting ESVs/cell (D) and the percentage of ESV positive cells (E) within the encysting cell population using monoclonal anti-CWP1 antibody for ESV detection. The data represents three biological replicates with the median of the ESV/cell distribution denoted by horizontal bars (D) and error bars representing the standard deviation (E). (F) ESVs were visualized using super resolution microscopy. Cells were induced to encyst for 22 h and ESVs and nuclear DNA were labeled using an anti-CWP1 antibody (green) and DAPI (blue) respectively. Maximum intensity projections (MIP) of image stacks are shown and the scale bar denotes a distance of 10 μm.
Fig 2.
Developmental regulation of the transcriptome along the trajectory of encystation.
(A) Non-clustering heatmap of all genes with FPKM ≥50 displaying a fold change ≥2 at any time along the encystation (n = 3106). Yellow and blue indicate up- and down regulated genes respectively on a Log2 scale. The cascade fashion of the global transcriptional changes together with similar periodicity for known and putative transcription factors and repressors (B) and chromatin modifiers (C) suggest a coordinated regulation of encystation on both the level of DNA accessibility and transcriptional activation. (D and E) Venn diagrams describing the numbers of unique and shared up- and down-regulated genes for the different time points post induction of cyst formation compared to non-induced trophozoites. The majority of transcriptional changes occur late in the differentiation (22h and cysts) with the majority of genes encoding hypothetical proteins.
Table 1.
Functional annotation cluster analysis of differentially expressed genes using DAVID.
Fig 3.
Localizations of epitope tagged proteins of genes in consensus between transcriptional studies.
Proteins tagged with HA or 3xHA (red) were visualized in conjunction with CWP1 (green) and DAPI stained DNA (blue) for trophozoites, 7 and 22 h post induction of encystation and cysts. (A) The hypothetical protein 10552-3xHA displayed an ER-like localization in a punctuate manner at both 7 and 22 h. (B) 12082-3xHA appears in vesicle like compartments in early encysting cells and ER like localization in late encysting cells. (C) 102813-3xHA is annotated as a Protein 21.1 and localizes to the nuclei in a proportion of the cells in the population, but not in cysts. (D) 103785-HA localizes to the ER and non-ESV, vesicle-like structures during encystation and in cysts. Scale bars represent 10 μm.
Fig 4.
Transcription of VSPs switch during differentiation.
Non-clustering heatmap of the highest transcribed VSPs during differentiation reveal changes in VSP expression along the trajectory of encystation. Elevations in abundance of several VSP transcripts not expressed in the starting population are observed at the different time points. The VSP with the highest cumulative level of expression in the population throughout encystation was GL50803_113797 (bolded) also known as TSA 417.
Fig 5.
Expression and localizations of epitope tagged proteins up-regulated in the late phase of encystation.
Proteins of genes with a transcriptional profile of late induction during encystation were epitope tagged with HA (red) and localized with CWP1 (green) and DAPI stained DNA (blue) by immunofluorescence. Scale bars represent 10μm. (A) 3731-HA localizes to the nuclei of a proportion of encysting cells and cysts. (B) 4984-HA showed localization to an unknown structure in mature cysts and appeared vesicle-like in encysting cells. (C) 23439-HA localized to vesicle-like structures in encysting cells and close to the membrane of the excyzoite in cysts. (D-F) Analysis of corresponding protein levels in extracts from trophozoites, 22h post induction and cysts by western blots reveal a marked increase in the 22.8 kDA 3731-HA (D), 23.6 kDa 4984-HA (E) and 10.8 kDa 23439-HA (F) over time. The larger band for 23439-HA indicates a potential association of the protein to the cyst wall.
Fig 6.
Summarizing image showing the transformation from the motile trophozoite via encyzoite to the final cyst stage.
The trophozoite monitors the external environment and encystation is induced via intracellular pathways that remain largely unknown. The cell passes a “point of no return” during early encystation after which it is no longer possible to relapse to the proliferating stage. Transcription factors (e.g. Myb2) activates encystation-specific genes among them are cyst wall proteins (CWP1-3). An overall increase of translation could be observed early in encystation as the production of CWPs is dramatically increased and the transportation in encystation vesicles (ESVs) begins. The vesicles undergo maturation steps after leaving the ER. The other component of the cyst wall, the UDP-GalNAc sugar (giardin), is also synthesized and secreted via encystation positive carbohydrate vesicles (ECVs). The enzymes involved in giardin synthesis are induced during encystation. During late encystation, the cell changes shape as it enters dormancy and the ventral disc together with the flagella are disassembled as the construction of the cyst wall proceeds. But the mechanism behind the assembly is still unknown. Often pre-cyst stages with a “tail” can be observed in encystation. Two rounds of DNA replication occur without cytokinesis rendering a cyst with four nuclei each with the genome ploidy of 4N. Interconnections between the nuclei in the cysts are formed and genetic material can be exchanged through the process “diplomixis”. During excystation, each cell receives one pair of non-sister nuclei (indicated as red and blue).