Givaudan-Roure Lecture - Association for Chemoreception Sciences

expression.
132 Slide [ ] Odorant Receptors & Transduction
REGULATION OF ODORANT RECEPTOR EXPRESSION
Reed R.R. 1, Lewcock J.W. 2 1Molecular Biology and Genetics,
HHMI/Johns Hopkins University, Baltimore, MD; 2Molecular Biology
and Genetics, Johns Hopkins University, Baltimore, MD
The expression of a single odorant receptor (OR) in each olfactory
neuron from a repertoire of more than 1000 genes is essential for odor
coding and axonal targeting. The genes encoding these receptors each
possess a simple genomic structure and in several cases, small DNA
segments surrounding the transcription initiation sites are sufficient to
direct expression of reporters in a pattern that mimics the endogenous
genes. One remarkable aspect of this gene regulation is that each
olfactory neuron expresses OR protein from only one allele of this
large, dispersed gene family. We have used targeted transgenesis,
insertion of defined DNA constructs at specific location in the genome
to identify a new role for OR protein as an essential regulator in the
establishment of mono-allelic OR expression. OR-promoter driven
reporters expresses in a receptor-like pattern, but unlike a native OR,
are co-expressed with an additional OR allele. Expression of a
functional OR from the identical promoter eliminates expression of
other OR alleles. The presence of an untranslatable OR coding
sequence in the mRNA is insufficient to exclude expression of a second
OR. Together, these data identify the OR protein as a critical element in
a feedback pathway that regulates odorant receptor selection. Current
efforts in the laboratory are focused on elucidating the nature of the
feedback signal and the mechanisms that lead to selective receptor
expression.
Supported by grants from NIDCD to R. R.
133 Symposium [ ] The Ins and Outs of Sensory Cilia
WHAT THE CHLAMYDOMONAS FLAGELLUM IS
TEACHING US ABOUT SENSORY CILIA
Witman G.B. 1 1Cell Biology, University of Massachusetts Medical
School (Worcester), Worcester, MA
Both motile cilia and non-motile cilia, including mammalian primary
cilia, are sensory organelles that display receptors and relay signals
about the extracellular environment to the cell body. The unicellular,
biflagellate green alga Chlamydomonas reinhardtii has provided an
essential foundation for understanding this important ciliary function.
A notable example is the recent discovery and characterization of
intraflagellar transport (IFT) in Chlamydomonas. IFT is a process in
which flagellar precursors are actively moved into the flagellum and out
to its tip, where axonemal assembly occurs; disruption of this process
blocks flagellar assembly. Genetic and biochemical studies in
Chlamydomonas have elucidated the molecular motors and other
components of the IFT system; all of these proteins have homologues in
other ciliated organisms, including C. elegans, D. melanogaster, and
mammals. Because IFT is necessary for ciliary assembly, mutation of a
gene encoding a mouse homologue of a Chlamydomonas IFT protein
disrupts assembly of primary cilia, providing a valuable tool for testing
the function of these widespread organelles (see abstract by G. Pazour).
Although primary cilia cannot be isolated, Chlamydomonas flagella can
be readily purified in amounts sufficient for biochemical analysis. An
ongoing proteomic analysis of the Chlamydomonas flagellum is
revealing numerous conserved proteins that are likely to be involved in
sensory processes. The mammalian homologues of these proteins are
prime candidates for carrying out sensory reception and signal
transduction in the primary cilium. Supported by NIH GM 30626.
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134 Symposium [ ] The Ins and Outs of Sensory Cilia
PROBING THE FUNCTION OF MAMMALIAN PRIMARY
CILIA BY ANALYSIS OF THE TG737 MOUSE
Pazour G.J. 1 1Molecular Medicine, University of Massachusetts
Medical School (Worcester), Worcester, MA
Most mammalian cells have a non-motile primary cilium projecting
from their surface. The importance of these organelles to mammalian
health and development has been highlighted by recent analysis of the
Tg737 mouse. This mouse has a mutation in the gene encoding the
IFT88 subunit of the intraflagellar transport particle (Pazour et al., J.
Cell Biol. 151:709-718) and develops polycystic kidney disease (PKD)
(Moyer et al., Science 262:1329-1333) and other disorders. The Tg737
mutation causes ciliary assembly defects in the kidney and other organs.
These ciliary defects are likely to be the primary cause underlying the
PKD and other diseases seen in the animal.
We hypothesize that primary cilia are serving as organizing centers
for sensory receptors and signaling proteins. In support of this, we and
others have shown that the polycystins are localized on kidney primary
cilia. The polycystins are membrane proteins thought to sense the state
of the kidney epithelium and control proliferation and differentiation of
these cells. Polycystin mutations cause excess cell proliferation in
kidney nephrons resulting in adult onset PKD in humans. The ciliary
assembly defect probably causes PKD by disrupting the localization of
the polycystins.
It is likely that all primary cilia serve similar functions in organizing
receptors and pathways to monitor extracellular parameters that are
important to the cell´s physiology.
Work in my laboratory is funded by the NIH (GM60992) and the
Worcester Foundation for Biomedical Research.
135 Symposium [ ] The Ins and Outs of Sensory Cilia
INTRAFLAGELLAR TRANSPORT MOTORS
Scholey J.M. 1, Ou G. 1, Snow J. 1, Gunnarson A. 1 1Center for Genetics
and Development and Section of Molecular and Cellular Biology,
University of California, Davis, Davis, CA
The assembly and function of sensory cilia depends upon
intraflagellar transport (IFT), the bidirectional transport of
macromolecular complexes called IFT-particles, along axonemal
microtubules (MTs) between the base of the cilium and the distal tip.
We are studying the role of IFT in the formation and function of the
sensory cilia on the endings of chemosensory neurons within the
nematode, C. elegans. These cilia serve as specialized compartments for
concentrating the sensory signaling machinery that detects chemical
cues in the environment and thereby play important roles in
chemosensory behaviour. Our hypothesis is that IFT-particles
contribute to ciliary function by carrying key components of the ciliary
axoneme, the signaling machinery, and possibly signals themselves,
between the base and the tip of the cilium, and that the transport of
these particles depends upon the action of two anterograde motors,
kinesin-II and OSM-3-kinesin and a retrograde motor, IFT-dynein. We
will report our recent progress in using light microscopy, biochemistry,
genomics and genetics to dissect the protein machinery that drives IFT
in this system.
Reference: Scholey JM, 2003, Intraflagellar Transport. Ann Rev.
Cell Dev. Biol., 19, 423.