Mechanisms contributing to the generation and maintenance of ciliary structural diversity in C. elegans.
Cilia and flagella are evolutionarily conserved microtubular organelles present in most organisms, and are assembled via intraflagellar transport (IFT) of ciliary precursors. In addition to the well-known motile cilia, most, if not all cells in metazoans have a non-motile primary cilia. These primary cilia act as cellular antennae, and play roles in sensing and transducing environmental signals. Individual cell types can elaborate morphologically diverse cilia; for example vertebrate olfactory neurons have multiple elongated cilia emanating from a dendritic knob, whereas the rod and cone photoreceptors exhibit elaborate outer segments that house the phototransduction machinery. Although these diverse ciliary structures are critical for the specialized functions of the cell type, the mechanisms that generate ciliary diversity are unknown.
C. elegans is an excellent model system in which to investigate the mechanisms required for the generation of ciliary morphological diversity. Eleven pairs of chemosensory neurons in the bilateral amphid organs of the head contain structurally diverse cilia. Eight of these sensory neuron types respond to aqueous attractants and contain relatively simple cilia structures consisting of one or two branches extending from the transition zone (channel cilia). Three additional neuron types, AWA, AWB and AWC, respond primarily to volatile odorants, and contain cilia that are morphologically unique and more elaborate (olfactory wing cilia) (Fig. 1). The molecular motors and components mediating IFT are well conserved in C. elegans. The goal of my project is to understand how these core ciliary building blocks are modified to generate and maintain specialized cilia structures.
1. Distinct Intra Flagellar Transport (IFT) mechanisms contribute to the generation of ciliary structural diversity in C. elegans
We have found that IFT mechanisms are deployed in a distinct manner in different cell types. To examine IFT in individual cilia types, we expressed IFT component genes and motors specifically in individual neurons. We first compared IFT in the ASH/ASI channel cilia and the AWB wing cilia. In the ASH/ASI cilia, the middle segments are built by the concerted action of the kinesin-II and the OSM-3 motors, whereas the distal segments are built by the OSM-3 motor alone (Snow et al., 2004). We found that unlike in the ASH/ASI cilia, the OSM-3 kinesin moves independently of the kinesin-II motor in the AWB cilia. Moreover, although OSM-3 is essential to extend the distal segments of the ASH/ASI cilia, it is not required to build the AWB distal segments (Fig. 2). We also identified a forkhead domain transcription factor (FKH-2) required for the specification of AWB ciliary morphology. We showed that FKH-2 regulates kinesin-II subunit gene expression specifically in AWB. Our results suggest that cell-specific regulation of IFT contributes to the generation of ciliary diversity, and provide insights into the networks coupling the acquisition of ciliary specializations with other aspects of cell fate. This work has now been published (Mukhopadhyay et al., 2007).
2. Signaling molecules regulate olfactory cilia structure in C. elegans.
In addition to mediating motility, cilia and flagella also act in sensory signaling, and signal transduction complexes required for responses to the environment are localized to these organelles. Mutations in signaling molecules involved in the vertebrate phototransduction cascade, including rhodopsin and cyclic nucleotide-gated channels, result in degeneration of the outer segments and photoreceptor apoptosis. Thus, mutations in these genes have been implicated in retinal degeneration disorders such as retinitis pigmentosa. The role of these signaling proteins and their interplay with IFT in regulating cilia structure and function is unclear.
Many genes implicated in olfactory signal transduction in the AWB neurons have been identified. We found that mutations in genes encoding the guanylyl cyclase odr-1 and the cyclic-nucleotide gated cation channel subunits tax-2 and tax-4 dramatically alter the structure of the AWB cilia. Temperature-shift experiments using a temperature-sensitive allele of tax-2 suggest that TAX-2 function is required in later larval stages to maintain AWB ciliary structure (Fig. 3). Rescue experiments with cell-specific expression of tax-2 suggest that TAX-2 acts cell-autonomously to regulate AWB ciliary structure. Intriguingly, we have found that the ciliary defects of odr-1and tax-4 mutants are suppressed by mutations in the anterograde IFT motor Kinesin-2 subunits klp-11 and kap-1, but not by mutations in the other anterograde IFT motor OSM-3. These results suggest that the alteration in cilia structures may be specifically dependent on Kinesin-2 mediated IFT. In addition, modulation of intracellular calcium signaling can also suppress the ciliary defects of signaling gene mutants. Although we also observe AWC olfactory cilia defects in both odr-1 and tax-2/tax-4 mutants, the channel cilia (some of which also express these signaling genes) appear unperturbed. These results indicate that signaling molecules required for AWB/C-mediated olfactory signal transduction also play a role in maintaining cilia structure, and that this function may be cell type-specific. Thus, we think that this system provides an excellent model in which to explore how mutations in signaling genes result in altered cilia structures, thereby leading to celluar dysfunction and death as in retinal degeneration disorders.
