Sunter, J. | bioRxiv Science

bioRxiv2020

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division. Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

cell biology↗

bioRxiv2019

A key regulatory protein for flagellum length control in stable flagella

Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved. A grow-and-lock model was proposed for the maintenance of stable flagella where a molecular lock is applied to prevent flagellum length change after assembly [5]. The molecular mechanisms of how this lock operates are unknown, but could be important in cells where an existing flagellum must be maintained whilst a new flagellum assembles. Here we show that Cep164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. It is only localised on the transition fibres of basal bodies of fully assembled flagella and missing from assembling flagella. In fact, basal bodies only acquire Cep164C in the third cell cycle after they assemble in trypanosomes. Depletion leads to dysregulation of flagellum growth with both longer and shorter flagella; consistent with defects in a flagellum locking mechanism. By controlling delivery of components into the old assembled flagellum, maintenance of stable flagella can occur but limits further growth. This offers an important explanation for how many eukaryotic unicellular cells maintain their existing flagella whilst growing new ones before these cells divide. This work also reveals additional regulatory roles for Cep164 in eukaryotic organisms.

cell biology↗