Dissecting the molecular mechanism of chromosome segregation in the marine plankton Diplonema papillatum

We are interested in discovering the fundamental principle of chromosome segregation in eukaryotes. Our focus is on the kinetochore, a highly complicated macromolecular protein machine that assembles onto centromeric DNA and interacts with spindle microtubules during mitosis and meiosis. Despite the essentiality of kinetochores for the survival of all present-day eukaryotes, there are at least two kinetochore systems with distinct compositions (1). In this project, we aim to identify the third type of kinetochores by studying diplonemids.

Diplonemids are diverse and abundant marine protists that are highly divergent from traditional model eukaryotes (2). They belong to the phylum Euglenozoa, which also includes euglenids and kinetoplastids. While euglenids have kinetochore proteins that are conserved in most eukaryotes, kinetoplastids have an unconventional kinetochore system. It remains unknown what kind of kinetochore proteins are present in diplonemids (3).

So far, very little is known about the mechanism of chromosome segregation or cell division in diplonemids. Diplonema papillatum is a model diplonemid for which a publicly available genome and molecular tools are available (2). We recently started characterizing its mitotic mechanism by examining the localization of conserved mitotic proteins by fluorescence microscopy (3).

The aim of this PhD project is to identify kinetochore proteins in Diplonema papillatum. This will be achieved by performing immunoprecipitation of conserved mitotic proteins that may interact with kinetochore proteins, followed by the identification of their interaction partners by mass spectrometry. Identified candidate proteins will be examined by epitope tagging and fluorescence microscopy as well as computational methods (e.g. AlphaFold), protein biochemistry and structural biology. We will also aim to develop molecular tools that will allow depletion of proteins in Diplonema papillatum. By identifying and characterizing a novel kinetochore system, we aim to reveal fundamental requirements and principles of eukaryotic chromosome segregation machinery.