A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Abstract

The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo-and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.T rypanosoma brucei is an important parasite of humans and domestic animals in sub-Saharan Africa, as the causative agent of sleeping sickness and nagana, respectively. Its complex life cycle involves transitions between tsetse fly vectors (its definitive hosts) and mammalian intermediate hosts. This life cycle involves a number of different cell stages, of which the procyclic form (found in the tsetse fly) and the slender bloodstream form (BSF) (found in the mammalian bloodstream) are the best studied in a laboratory setting. The procyclic form and the BSF of T. brucei share similar cytoskeletal architectures (1, 2).The principal feature of this cytoskeleton is a corset of microtubules that lie directly underneath the plasma membrane and impart to the cell its distinctive shape (3). A single invagination of the plasma membrane, termed the flagellar pocket (FP), constitutes a distinct subdomain and is found at the posterior end of the cell (4). The FP is the site of all endo-and exocytic traffic (5, 6). Abutting the FP membrane is a basal body that nucleates the single flagellum of the trypanosome cell. The flagellum exits the FP and is adhered longitudinally to the cell body along a left-handed helical path (7). Once outside the FP, the axoneme of the flagellum is paralleled by an associated intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of a paracrystalline lattice and is associated with cellular motility (8). Nucleated adjacent to the basal body is a specialized microtubule quartet that traces around the FP and then underlies the flagellum as far as the anterior end of the cell (4).The small cylinder of membrane that connects the FP to the rest of the plasma membrane constitutes a third subdomain and is called the flagellar pocket neck (FPN) (4). A number of discrete cytoskeletal structures cluster around the FPN membrane on its cytoplasmic face. Of these, the best characterized is an electrondense horseshoe-shaped structure named th...

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

“…In this way, the contribution of other sequences or domains could be discounted. The MORN1 protein from the early-branching eukaryote Trypanosoma brucei is an ideal candidate in this regard, and has the advantage of also being well-characterised at a cell biology level (Morriswood, He et al, 2009, Esson, Morriswood et al, 2012, Morriswood, Havlicek et al, 2013, Morriswood & Schmidt, 2015).…”

“…In functional terms, depletion of TbMORN1 by RNAi in the mammalian-infective form of the parasite resulted in a lethal phenotype (Morriswood et al, 2009). Functional analysis indicated that the protein might be involved in endocytosis, as well as regulating the flow of macromolecular cargo through the neck of the flagellar pocket (Morriswood & Schmidt, 2015).…”

Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation

“…Cytoskeleton- and flagella-enriched fractions were prepared following a published protocol5960 with minor modifications. Briefly, 1×10 8 cells were harvested by centrifugation (1500 × g, 10 min, 4 °C) and washed in 1 ml PEME buffer (2 mM EGTA, 1 mM MgSO4, 0.1 mM EDTA, 0.1 M piperazine-N,N′-bis (2-ethanesulfonic acid)-NaOH (PIPES-NaOH), pH 6.9).…”

Morphological and Functional Aspects of Cytoskeleton of Trypanosomatids

“…The bilobe is located near the Golgi apparatus; it is thought to function in Golgi apparatus replication and as an adaptor during cytokinesis [34]. Recently, TbMORN1 depletion experiments suggested that this protein allows access of endocytic tracers into the flagellar pocket [35].…”