Abstract
Flagellated cells are of great evolutionary importance across animal and plant species. Unlike higher plants, flagellated cells are involved in reproduction of macro-algae as well as in early diverging land plants. Euglena gracilis is an emerging flagellated model organism. The current study reports that a specific calmodulin (CaM2) involved in gravitaxis of E. gracilis interacts with an evolutionary conserved flagellar protein, EgPCDUF4201. The subsequent molecular analysis showed clearly that EgPCDUF4201 is also involved in gravitaxis. We performed subcellular localization of CaM2 using immunoblotting and indirect immunofluorescence. By employing yeast two-hybrid screen, EgPCDUF4201 was identified as an interaction partner of CaM2. The C-terminus of EgPCDUF4201 is responsible for the interaction with CaM2. Silencing of N- and C-terminus of EgPCDUF4201 using RNAi resulted in an impaired gravitaxis. Moreover, indirect immunofluorescence assay showed that EgPCDUF4201 is a flagella associated protein. The current study specifically addressed some important questions regarding the signal transduction chain of gravitaxis in E. gracilis. Besides the fact that it improved the current understanding of gravity sensing mechanisms in E. gracilis, it also gave rise to several interesting research questions regarding the function of the domain of unknown function 4201 in flagellated cells.
Highlights
Euglena gracilis is a photosynthetic, eukaryotic unicellular organism which is a member of the Euglenozoa along with the parasites of the genera Trypanozoma and Leishmania[1]
calmodulin 2 protein (CaM2) is present in the cell body and the flagella of E. gracilis
In order to better understand the function of CaM2, we aimed to identify its subcellular localization by indirect immunofluorescence (IIF) and cell fractionation followed by Western blot, using anti CaM2 genomic antibody
Summary
Euglena gracilis is a photosynthetic, eukaryotic unicellular organism which is a member of the Euglenozoa along with the parasites of the genera Trypanozoma and Leishmania[1]. As shown under laboratory standard conditions cells show a transition from positive to negative gravitaxis as culture grows from young to old respectively It has been demonstrated, in parabolic flight conditions (transition from hyper g to micro g as well as from micro g to hyper g), that this orientation is an active physiological process in which the beating of the flagella is involved and controlled by gravity[6]. Additional studies are required in this direction as several putative stretch sensitive/mechanosensitive channels have been identified in the mRNA library of E. gracilis (unpublished data) Besides this open question regarding the exact nature of gravitaxis specific SSCIC, a reasonable progress has been made regarding the underlying molecular mechanism of gravitaxis in E. gracilis. The resulting binding partner was further characterized by heterologous expression, in silico analysis and its subcellular localization was analyzed
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