The single flagellum of Trypanosoma brucei has been unveiled as a key player in determining cell polarity, division, size and shape.
The flagellum, an appendage whose structure is conserved from protists to mammals, extends from the cell body. It is assembled by adding subunits to the distal tip. However, the flagellum lacks the machinery for protein synthesis and a transport system for subunits synthesized in the cell body is needed. A specialized system, known as intraflagellar transport (IFT), delivers 'rafts' of proteins that are synthesised in the cell body to the flagella tip, and can also transport proteins from the flagellum back to the cell body. Motor proteins drive movement of rafts, which contain additional IFT proteins known as cargoes. Defects in IFT prevent flagellum formation in most eukaryotes tested so far. In new research published in the EMBO Journal RNAi and cell biology were combined to probe the function of the trypanosome flagellum.
Kohl et al. mined T. brucei genome sequences for putative IFT homologues. From several homologues identified, two genes were selected. One gene encodes a putative cargo protein whilst the other gene encodes a putative motor protein. By coupling gene silencing using RNAi with microscopy, Kohl et al. showed that separate silencing of either the putative motor or cargo homologue resulted in progressive flagellum shortening. A shortened flagellum is initially produced because RNAi results in a dwindling pool of IFT proteins, which in turn reduces transport of the proteins needed for flagellum assembly. Eventually daughter cells had no flagellum. So, IFT is functional in T. brucei and is required for flagellum assembly and for control of flagellum length. Mutant cells lacking a flagellum lost polarity, as demonstrated by mislocalization of clathrin, a typical cell organization marker. These mutants also lost their distinctive shape and had no clear anterior or posterior end. So, both cell polarity and internal organization were lost when flagella assembly was disrupted.
During induction of RNAi, mutants not only produced a shortened flagellum but were smaller than wild-type cells. The length of the flagellum correlated with cell size — the shorter the flagellum, the smaller the daughter cell. Before cell division, a new flagellum is assembled alongside the old flagellum. By monitoring mutants during induction of RNAi the authors deduced that the tip of the newly synthesised flagellum defines the site of cytokinesis. Alongside the flagellum in the cytoplasm there is a flagellar attachment zone (FAZ) which also replicates before cell division. Mutant trypanosomes which completely lack flagella still have a FAZ but its shape is shortened and irregular. Since these cells can still divide, the FAZ is the best candidate for a marker of cytokinesis.
During the life cycle of T. brucei, there are several morphologically distinct cell-types. It's possible that regulating IFT — and therefore regulating flagellum length — could be used to modulate parasite differentiation.
ORIGINAL RESEARCH PAPER
Kohl et al. Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes. EMBO J. 22, 5336–5345 (2003)
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Jones, S. Trypanosome flagellum — motility and more. Nat Rev Microbiol 1, 172 (2003). https://doi.org/10.1038/nrmicro792