The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Abstract

Cilia and flagella are assembled by intraflagellar transport (IFT). The protist Trypanosoma brucei is used to investigate retrograde IFT. A model is proposed by which the IFT dynein particle is assembled in the cytoplasm, transferred to the base of the flagellum, and associated with the IFT machinery in a manner dependent on the IFT-A complex.

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length

“…For IFT140 RNAi cells, it was 2.66± 0.89 and 0.79/s (n=233). These values are within the usual range for IFT in trypanosomes (Bhogaraju et al, 2013;Blisnick et al, 2014;Buisson et al, 2013;Huet et al, 2014). In contrast, observation of live IFT88…”

“…To firmly correlate the modification in abundance of the electrondense structures with IFT, an immunofluorescence assay (IFA) was performed on methanol-fixed trypanosomes, using double labelling with MAb25, a monoclonal antibody that recognizes a protein found all along the axoneme (Pradel et al, 2006) and with a monoclonal antibody raised against IFT172, a classic IFT marker (Absalon et al, 2008;Blisnick et al, 2014). MAb25 produced staining along the whole length of the flagellum in both mature flagella (white arrows) and flagella undergoing construction (yellow arrows, Fig.…”

“…IFT has been imaged by tagging multiple IFT-B or dynein subunits and is active during both construction and maintenance. The expression of various components of the IFT-A and IFT-B complexes has been individually silenced using inducible RNA interference (RNAi), resulting in failure of flagellum construction (Absalon et al, 2008;Adhiambo et al, 2009;Blisnick et al, 2014;Davidge et al, 2006;Franklin and Ullu, 2010;Huet et al, 2014;Kohl et al, 2003). Usually, inhibition of IFT-B proteins (for example IFT88) blocks axoneme construction in agreement with a default in anterograde transport, whereas absence of IFT-A proteins (such as IFT140) leads to formation of short flagella filled with IFT material, indicative of defects in retrograde transport (Absalon et al, 2008).…”

Imaging intraflagellar transport in trypanosomes

“…The pZJM vector containing a fragment of a gene of interest cloned between these promoters can be transfected in the 29-13 cell line, allowing for tetracyclineinducible RNAi (Wang, Morris, Drew, & Englund, 2000). This approach has been highly valuable to demonstrate the role of multiple IFT genes in flagellum construction (Absalon et al, 2008;Adhiambo et al, 2009;Blisnick et al, 2014;Davidge et al, 2006;Franklin & Ullu, 2010;Huet et al, 2014;Kohl et al, 2003). Such IFT RNAi cell lines can be transformed with a second plasmid, with a different drug resistance gene, allowing expression of an IFT fusion protein.…”

“…To avoid potential side effects of overexpression, a specific expression vector relying on suitable upstream and downstream regulation sequences adapted to flagellar genes was used (Adhiambo, Blisnick, Toutirais, Delannoy, & Bastin, 2009;Huet, Blisnick, Perrot, & Bastin, 2014). More recently, endogenous tagging has become the method of choice to tag IFT proteins with a fluorescent marker (Bhogaraju et al, 2013;Blisnick et al, 2014). We will review here these different tagging approaches and describe the methods used to record IFT.…”