Tools for analyzing intraflagellar transport in trypanosomes.
African trypanosomes are evolutionary-divergent eukaryotes responsible for sleeping sickness. They duplicate their single flagellum while maintaining the old one, providing a unique model to examine mature and elongating flagella in the same cell. Like in most eukaryotes, the trypanosome flagellum is constructed by addition of novel subunits at its distal end via the action of intraflagellar transport (IFT). Almost all genes encoding IFT proteins and motors are conserved in trypanosomes and related species, with only a few exceptions. A dozen of IFT genes have been functionally investigated in this organism, thanks to the potent reverse genetic tools available. Several alternative techniques to trigger RNAi are accessible, either transient RNAi by transfection of long double-stranded RNA or by generation of clonal cell lines able to express long double-stranded RNA under the control of tetracycline-inducible promoters. In addition, we provide a series of techniques to investigate cellular phenotypes in trypanosomes where expression of IFT genes has been silenced. In this chapter, we describe different methods for tagging and expression of IFT proteins in trypanosomes and for visualizing IFT in live cells.
Julkowska
Daria
D
Trypanosome Cell Biology Unit, Institut Pasteur and CNRS, Paris, France.
Bastin
Philippe
P
D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D005407:Flagella / Q000378:metabolism* / Q000648:ultrastructure; D020868:Gene Silencing; D005822:Genetic Vectors; D018503:Genome, Protozoan; D007150:Immunohistochemistry / Q000295:instrumentation / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure*
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins