Functional genomics of intraflagellar transport-associated proteins in C. elegans.
The nematode Caenorhabditis elegans presents numerous advantages for the identification and molecular analysis of intraflagellar transport (IFT)-associated proteins, which play a critical role in the formation of cilia. Many proteins were first described as participating in IFT in this organism, including IFTA-1 (IFT121), DYF-1 (fleer/IFT70), DYF-2 (IFT144), DYF-3 (Qilin), DYF-11 (MIP-T3/IFT54), DYF-13, XBX-1 (dynein light intermediate chain), XBX-2 (dynein light chain), CHE-13 (IFT57/HIPPI), orthologs of Bardet-Biedl syndrome proteins, and potential regulatory protein, IFTA-2 (RABL5/IFT22). Transgenic animals bearing green fluorescent protein (GFP)-tagged proteins can be generated with ease, and in vivo imaging of IFT in both wild-type and cilia mutant strains can be performed quickly. The analyses permit detailed information on the localization and dynamic properties (velocities along the ciliary axoneme) of the relevant proteins, providing insights into their potential functions in processes such as anterograde and retrograde transport and cilium formation, as well as association with distinct modules of the IFT machinery (e.g., IFT subcomplexes A or B). Behavioral studies of the corresponding IFT-associated gene mutants further enable an understanding of the ciliary role of the proteins-e.g., in chemosensation, lipid homeostasis, lifespan control, and signaling-in a multicellular animal. In this chapter, we discuss how C. elegans can be used for the identification and characterization of IFT-associated proteins, focusing on methods for the generation of GFP-tagged IFT reporter strains, time-lapse microscopy, and IFT rate measurements.
Inglis
Peter N
PN
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby BC V5A1S6, Canada.
Blacque
Oliver E
OE
Leroux
Michel R
MR
D000818:Animals; D056444:Axonemal Dyneins / Q000235:genetics / Q000378:metabolism; D054468:Axoneme / Q000378:metabolism / Q000648:ultrastructure; D001692:Biological Transport / Q000502:physiology; D017173:Caenorhabditis elegans / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D005407:Flagella / Q000378:metabolism* / Q000648:ultrastructure; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000166:cytology / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D056444:Axonemal Dyneins; D016547:Kinesins