The Functional Characterization of TcMyoF Implicates a Family of Cytostome-Cytopharynx Targeted Myosins as Integral to the Endocytic Machinery of Trypanosoma cruzi

Figures

• 

  FIG 1. (Top panel) Bioinformatic identification of potential SPC proteins. The list of 17,713 annotated T. cruzi genes from the Y strain genome assembly was narrowed down to 9,612 genes that do not have orthologues outside the Euglena genus. This list was further trimmed to 4,265 genes by eliminating all genes that have orthologues in Leishmania and T. brucei which lack the SPC. The final filter eliminated genes that did not have orthologues in the monoxenous kinetoplastid parasite Paratrypanosoma confusum, which has an SPC, resulting in a list of 217 potential SPC genes. (Bottom panel) List of seven proteins that have been shown to localize to the SPC. ID, identifier.

• 

  FIG 2. Identification and localization of six SPC proteins. (A) CP4 localizes to the SPC, potentially on the CyQ and MtQ (arrow) microtubule root fibers and the basal bodies associated with the flagellum (arrowhead). (B to D) CP5 (B), CP6 (C), and CP7 (D) localize to the POR of the SPC labeled by the lectin concanavalin A (purple arrows). CP5 also shows basal body localization (arrowhead). (E and F) Two proteins annotated to contain myosin motor domains (MyoF [E] and MyHd [F]) localize to the cytopharynx of the SPC. Nuclei and kinetoplasts in all fluorescent images were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bars, 2 μM.

• 

  FIG 3. Dominant-negative MyoF mutants are defective in SPC endocytosis. (A) Schematic of the MyoF gene showing the annotated myosin and SCP1 coiled-coil domains. (B) I-TASSER structure prediction of MyoF (colors) aligned with a known myosin crystal structure (gray) showing ATP binding P-loop region (green) and switch regions (magenta). A conserved lysine residue that is essential for P-loop function is shown in red (K198, arrow). (C) Sequence of the ATP binding P-loop region showing the K198 amino acid residue (red) that was mutated to a glutamine (K198Q) to generate a rigor mutant of MyoF (MyoF-K198Q) for dominant-negative expression. (D) Immunoblot probed with anti-Ty antibody showing similar levels of MyoF and MyoF-K198Q in the chosen subclones. LC, loading control. (E and F) Overexpressed MyoF (E) and MyoF-K198Q (F) fused to mNeon exhibited the same localization in epimastigotes. (G and H) Flow cytometry of epimastigotes after fluorescent BSA feeding shows that the actin inhibitor cytochalasin D prevents SPC endocytosis. (I and J) Flow cytometry of epimastigotes after fluorescent BSA feeding shows a dominant-negative endocytosis defect in the MyoF-K198Q mutants. G and I, counts; H…

• 

  FIG 4. MyoF deletion mutants exhibit a reduction in the rate of SPC endocytosis. (A) Scheme for CRISPR/Cas9 gene replacement and complementation strategy used to generate ΔMyoF deletion and complemented (Δ::MyoF-Ty) mutants. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) PCR amplification of the genomic locus showing replacement of both parental loci (high-molecular-weight [MW] band) with the blasticidin resistance gene (low-MW band) in a subclone of the ΔMyoF mutant. PCR of a nonsubcloned population of complemented parasites shows insertion of the MyoF-Ty-Hygro repair template into the MyoF locus. (C) Anti-Ty immunoblot of lysates from parental, ΔMyoF, and (Δ::MyoF-Ty) epimastigotes showing expression of MyoF-Ty in the complemented parasites. (D) Immunofluorescence assays of a (Δ::MyoF-Ty) epimastigote showing expression and proper SPC localization of MyoF-Ty. (E) Flow cytometry analysis of epimastigotes fed fluorescent BSA showing a reduced rate of feeding in ΔMyoF mutants, a phenotype that is partially rescued in the complemented (Δ::MyoF-Ty) mutants. (F) Quantification of the feeding rate results represented by reduced mean fluorescence of endocytosing epimastigotes. A…

• 

  FIG 5. Three additional T. cruzi myosins localize to the SPC. (A) Overexpression and superresolution structured illumination (SR-SIM) microscopy of MyoC-mNeon revealed cytopharynx localization, suggesting that MyoC may provide redundancy to the cytopharynx endocytic machinery. (B and C) MyoB (B) and MyoE (C) both localize to the preoral ridge of the SPC (purple arrows). Nuclei and kinetoplasts in all fluorescent images were stained with DAPI (blue). Scale bars, 2 μM.

• 

  FIG 6. The C-terminal tail regions of myosin F, C, B, and E are sufficient for SPC targeting. (A) Schematic of myosin genes and the regions (green and red lines) that were fused to the C terminus of mNeon for localization. The green-colored regions were sufficient for localization to the SPC when fused to mNeon, whereas the red-colored regions were not. (B, C, and D) The MyoF-Ct1 (B) and MyoF-Ct3 (D) regions were sufficient to target mNeon to the SPC cytopharynx, although MyoF-Ct3 had a notably less defined localization. MyoF-Ct2 (C) was not sufficient for SPC targeting but did demonstrate localization adjacent to the kinetoplast (arrowhead). (E and F) MyoC-Ct2 (F) was sufficient for targeting mNeon to the SPC cytopharynx, whereas MyoC-Ct1 (E) was not. (G and H) The C-terminal regions of MyoB (G) and MyoE (H) were both sufficient to target mNeon to the SPC preoral ridge. Nuclei and kinetoplasts in all fluorescent images were stained with DAPI (blue). Scale bars, 2 μM.

• 

  FIG 7. A family of orphan myosins target to distinct regions of the SPC. A cartoon schematic of a T. cruzi epimastigote is shown, highlighting the internal and external structures of the cytostome-cytopharynx complex. The microtubules of the axoneme, cytoskeleton, and root fibers are depicted in gray. The individual components of the SPC and parasite body are referenced in the associated key. Different colored lines denote the observed locations of the four orphan myosins targeted to distinct subregions of the cytostome-cytopharynx complex.