Project Summary
Flagella and the synonymous cilia protrude above the cell surface to detect signals and whip surrounding fluid.
These remarkable cellular antennae and propellers play central roles in human development and the
operations of most organs. Yet to grow and maintain the structural core supporting these hair-like organelles, a
unique train-like system termed intraflagellar transport (IFT) must continuously deliver a variety of proteins
made in the cell body to the distal tip of flagella. Defects in trafficking and the structure core underlies many
debilitating genetic disorders and chronic illnesses. While IFT has been investigated extensively, how and
where IFT trains load various core proteins remains elusive. Founded on technical breakthroughs in the single
cell protist, Chlamydomonas, which is amenable to diverse experimental approaches, preliminary data
provides an unprecedented opportunity to elucidate this complicated process and involved genes. A model
protein complex, the radial spoke, fails to assemble at the distal part of flagella in the mutant strains defective
in the ARMC2 gene or PF5 gene. Under a microscope, fluorescent ARMC2 and IFT trains are concentrated
near the flagellar entrance, whereas radial spokes are abundant and dynamic in a novel “cargo hub” in the cell
body. This proposal aims to test objectively and rigorously the hypothesis that the ARMC2-PF5 adaptor
facilitates and streamlines radial spoke trafficking from the cytoplasmic cargo hub to the flagellar distal tip for
assembly. Aim 1 will generate transgenic strains expressing ARMC2, radial spokes and IFT trains that are
tagged to fluorescent proteins of superior optical properties. Various imaging systems and quantitative analysis
will be used to reveal the locations of fluorescent molecules and to track their movements; and to determine
how growing or shrinking flagella affect the protein cargoes in the putative cargo hub. Aim 1 will further test if
the distal deficiency will be lessened by slowing ciliary growth rates to allow vestigial assembly to catch up.
Aim 2 will use sequencing, genetic and biochemical approaches to determine if the ARMC2 gene or PF5 gene
are identical or distinct; and to identify candidate ARMC2 interactors, such as radial spokes, IFT trains and
perhaps PF5 protein. Aim 3 will use molecular approaches to test whether the major segments in ARMC2 are
involved in binding IFT trains and radial spokes respectively. Together, the proposed projects will define the
new cargo hub and reveal the molecular basis that enables IFT trains pick up and drop off cargoes at proper
locations. The findings and innovative approaches will stimulate novel ideas in the ciliary field, empower
researchers facing similar challenges, and accelerate the discoveries of similar atypical flagellar genes that are
likely causative to cilia-related diseases. Modulation of relative assembly rates of individual complexes and the
entire flagellum will encourage the development of innovative therapeutic strategies. Finally, the combination of
classical experiments, cutting-edge technologies and creative problem-solving strategies will attract and
prepare graduate and many undergraduate students to pursue careers in the future biomedical fields.