Identification and Characterization of Golgin-63 of Trypanosoma cruzi

The Golgi Complex (GC) is composed of cisterns arranged in the form of stacks and is polarized, with cis-face and trans-face that communicates with the plasma membrane and compartments involved in the endocytosis and exocytosis processes. This arrangement is maintained by the presence of proteins, such as Golgins, the main proteins present in the GC of eukaryotes. These coil-like proteins are essential for the anchoring and coupling of transporters to the target membrane, membrane fusion, and structural support of GC cisterns. In T. cruzi, the GC is located close to the flagellar pocket and kinetoplast and presents morphology similar to that described in other organisms. However, there is still little understanding of its biogenesis and composition in T. cruzi. This study is to identify a GC cell marker in T. cruzi to analyze the biogenesis and structure of the organelle during the parasite's life cycle. For this, a search was conducted in the genome of T. cruzi for GC proteins, and Golgin 63 (TcG63) was selected. TcG63 was labeled with mNeonGreen and Myc (TcG63::mNG) using CRISPR/Cas9 system. The expression of TcG63::mNG was confirmed by flow cytometry by displacement of the peak in the mutant relative to the control and by immunodetection with the observation of the 95.8 kDa band corresponding to TcG63::mNG. We observed a ring-shaped marking in fluorescence microscopy, commonly seen in epimastigotes, trypomastigotes from cell culture, and amastigotes. This suggests a location at the ends of GC cells like that seen in other organisms. In metacyclic trypomastigote, we notice a compact point structure. As for the topology of the CG in the cell, we noticed that it accompanies the migration of the kinetoplast, becoming perpendicular to it in epimastigotes and amastigotes. In the trypomastigotes of cell culture and metacyclic forms, the GC is found between the nucleus and the kinetoplast.