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A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton
M M Baqui1, C S Takata, R V Milder
1Departamento de Parasitologia, Universidade de Säo Paulo, Brazil.
Abstract:
A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens. This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa. A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species. In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with [35S]methionine. This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin). Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton. Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex. We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.
Insights
A giant cytoskeleton protein, similar to titin, was identified in Phytomonas serpens. This protein is located at the anterior end, potentially linking the cell skeleton to the flagellum membrane.
Area of Science:
- Cell Biology
- Parasitology
- Biochemistry
Background:
- The cytoskeleton of flagellated protozoa plays crucial roles in cell structure and motility.
- Identifying novel protein components is essential for understanding parasite biology.
Purpose of the Study:
- To characterize a large, previously unidentified protein component of the Phytomonas serpens cytoskeleton.
- To investigate the localization and potential function of this giant protein.
Main Methods:
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular mass.
- Polyclonal antibody production and immunoblot analysis for protein detection.
- Metabolic labeling with [35S]methionine and immunoprecipitation.
- Indirect immunofluorescence microscopy and immunogold electron microscopy for localization.
Main Results:
- A megadalton protein, migrating as a 2500-3000 kDa doublet, was identified in Phytomonas serpens.
- An antibody against this protein reacted specifically with Phytomonas species but not other trypanosomatids or muscle titin.
- Microscopy revealed the protein caps the anterior microtubules at the junctional complex.
Conclusions:
- A novel giant protein is a component of the Phytomonas serpens cytoskeleton.
- This protein is localized at the anterior cell pole and may link the cytoskeleton to the flagellum.
- The findings provide insights into the structural organization of flagellated protozoa.