Development of a tool for GFP expression in live cells of the eukaryote Arcella intermedia, to determine the involvement of Rab-GTPases in the shell formation process

Abstract

Arcellinida (Amoebozoa) is a monophyletic group of testate amoebae characterized by the presence of a shell (test). How this lineage gained the capability to build a hardened outer structure remains unknown. To elucidate the evolutionary origin of arcellinids' shell, it is necessary to understand its formation process, which has been well-described for the genus Arcella based on cytomorphological data. Currently, we lack information about the molecular apparatus involved in shell formation. Based on the cytomorphological descriptions and recent results of the candidate's main PhD project, Rab8 and Rab11 subfamilies of the Rab GTPase gene family have become key candidates to be involved in the shell formation process. Here, we propose to establish and apply a protocol for functional expression of Rab-GFP (Green Fluorescent Protein) in Arcella intermedia, aiming to document the participation of Rab8 and Rab11 subfamilies in the shell formation process. Additionally, we aim to perform functional expression of Rab-GFP in other amoebozoans. Ultimately, the candidate's main PhD project and the present proposal enable us to shed light on the evolutionary origin of the Arcellinida shell, while establishing a model framework based on single-cell transcriptomics, phylogenetics, immunocytochemistry, and GFP suitable to be applied for other amoebozoans and micro-eukaryotic lineages.