Membrane domains and flagellar pocket boundaries are influenced by the cytoskeleton in African trypanosomes

Abstract

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell's sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.electron tomography ͉ endocytosis ͉ freeze fracture ͉ Trypanosoma brucei ͉ flagellum P arasitic protozoa, like many cells, organize their surface membranes into subdomains or microenvironments that are specialized to accomplish particular recognition, secretory, and endocytic functions. The surfaces of parasitic organisms, however, have an extra level of constraint; they must perform these vital roles while the organism avoids elimination by defensive responses mounted by the host.The compartmentalization of surface membrane is especially interesting in African trypanosomes because of their particular parasitic lifestyle. Trypanosoma brucei is a flagellate protozoan that lives in the blood of mammals in an exclusively extracellular form, fully exposed to the host immune system. Survival is assisted both by rapid endocytosis that clears the entire cell surface of bound antibodies within Ϸ7 min (1) and through the expression of a series of immunologically distinct cell surface coats. Each coat is produced from a single GPI-anchored variant surface glycoprotein (VSG; ref.2). Periodic switching of the expressed VSG gene from a vast silent library enables the parasite to avoid clearance by the host's adaptive immune response, hence prolonging infection and increasing the chances of transmission by the bite of a tsetse fly.African trypanosomes maintain the VSG coat free of most invariant endocytosis receptors that could otherwise elicit an immune response from which the organism could not escape. This is achieved through specialization of the plasma membrane at the base of the flagellum to create a protected invagination, the flagellar pocket (FP), in which the invariant receptors for endocytosis and innate immune evasion are concentrated (3, 4). The FP is the sole site for all endocytosis and exocytosis, and along with this critical function, it also plays important roles in protein and lipid sorting and recycling (for review, see ref. ...

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

“…Regardless of the number and orientation of the BSA molecules attached to the gold grain, the total particle size will be greater than that of ConA, and generally above 100 Å. Robust uptake of BSA-gold (5 nm and larger) by T. brucei has been demonstrated in the past (20,24). The size exclusion model predicts that it should be harder for the BSA-gold to enter following TbMORN1 depletion (Fig.…”

“…Third, is the neck channel still open following TbMORN1 depletion, or has it collapsed? The neck channel is a recently described corridor through the FPN that connects the FP lumen to the extracellular space (20). The position of this channel alongside the specialized microtubule quartet puts it in close proximity to the position of TbMORN1.…”

“…Parallel cultures of BSF TbMORN1 RNAi cells were seeded at 10 5 cells/ml in a total volume of 50 ml, and RNAi was induced in one culture by the addition of 1 g/ml of tetracycline. At the desired time points, samples of 7 ϫ 10 5 cells from each population were fixed using isothermal glutaraldehyde according to a previously published protocol (20). Fixed cells were washed with phosphate-buffered saline (PBS) and attached to poly-L-lysine-coated coverslips by centrifugation (1,000 ϫ g for 1 min).…”

The ciliary pocket: an endocytic membrane domain at the base of primary and motile cilia

“…6A,B), a basic marker for clathrin-dependent endocytosis. In contrast to Trypanosomatids, where budding takes place at a specific sub-localisation of the FP (Gadelha et al, 2009), CCPs are detected over the whole surface of the ciliary pocket (Fig. 4).…”

Polo-like kinase is necessary for flagellum inheritance in Trypanosoma brucei

“…The flagellum emerges from the membrane of the flagellar pocket and must therefore exit this compartment. The flagellar pocket collar (FPC), a structure comprising the protein BILBO1, tightly clinches the neck of the flagellar pocket against the flagellum, which limits access to the compartment Gadelha et al, 2009). The neck of the flagellar pocket is also partially encircled by the hook-like extension of a MORN1-containing cytoskeletal complex, the stem of which coincides with the posterior part of the FAZ (Morriswood et al, 2009).…”