Flagellar motility is widely distributed among eukaryotes. It is characteristic of many protists and the sperm cells of most plants and animals. Eukaryotic flagella are formed by basal bodies (kinetosomes), which are cylindrical structures of nine microtubular triplets. Basal bodies (and the structurally similar centrioles) are highly conserved and thought to function as templates for the microtubular system of the axoneme. The basal bodies are attached to a set of cytoskeletal elements which together with the basal bodies form the flagellar basal apparatus (mastigont). The basal body-associated structures include microtubular flagellar roots, fibers interconnecting basal bodies or connecting them with the plasma membrane or the cell nucleus, and fibers extending into the cytosol.
At the end of the last century, excavate protists were recognized as a monophyletic group primarily due to similar morphological features. A typical excavate structure implies flagellate unicellular organisms with a feeding groove, a place on the surface of the cell that is used for food intake, supported by a specific architecture of microtubular roots and associated fibers. Shortly after the definition of this group, hypotheses contradicting the monophyletic nature of these protists were proposed. Thanks to the accumulation of data on the ultrastructure of the flagellate apparatus of different groups of excavates, as well as confirmation in molecular phylogeny, we now assume that excavates represent a paraphyletic group from which all other eukaryotes evolved. Given that excavates generally possess one of the most elaborated mastigont and cytoskeletal apparati among eukaryotes, the interpretation of its individual elements represents the basis for understanding the evolutionary history of the last common eukaryotic ancestor - LECA.
In the proposed project, we will investigate the structure and ontogeny of mastigont in Paratrimastix pyriformis (Metamonada), the model organism in our laboratory. P. pyriformis is a free-living species characterized by a typical excavate morphology, i.e., suspension feeding groove and four flagella of which one is directed anteriorly (or antero-laterally), one posteriorly, and others laterally. The posterior flagellum possesses two vanes with thickened vane margins. So far, no data are available on the cell division of species from this genus and the role of cytoskeletal elements in this process. Although the ultrastructure of P. pyriformis and its relatives has been investigated for the past few decades, we want to go a step further and investigate the details of the process of cell division and ontogeny of this cytoskeleton.
The aim of the project is to describe the process of cell division in Paratrimastix pyriformis, with emphasis on the flagellar apparatus roles of basal bodies and the inheritance of the cytoskeletal elements throughout the cycle. We will use antibodies staining alpha-tubulin to visualise microtubular roots and in-house produce antibodies against striated fiber (SF)-assemblin which specifically stains roots connected to the posterior basal body. Four types of microscopy will be applied: scanning electron microscopy, transmission electron microscopy, fluorescence microscopy and expansion microscopy. With scanning electron microscopy, the morphological aspect of cell division will be visualized, while transmission electron microscopy will be used for visualization and ultrastructural analysis of basal bodies and their associated microtubular elements in different phases of cell division. Special attention will be paid to expansion microscopy, a powerful super-resolution method that allows nanoscale imaging of specifically labelled biological specimens using conventional microscopes.
Salary: co-founding 1000 EUR/month is ensured
Co-founding resources: GACR 23-07277S
Supervisor: Vladimír Hampl
Position available from: January 1, 2024
Deadline date for applications: July 27, 2023