Elizabeth F. Smith
Regulation of Eukaryotic Flagellar Assembly and Motility
Cilia and flagella are found on diverse cell types including sperm cells of vertebrates and some invertebrates, unicellular protozoa, and several vertebrate epithelial cell types. In mammals, for example, motile cilia found on cells lining the brain ventricles circulate cerebrospinal fluid; cilia in the respiratory tract sweep debris from the lungs; and oviduct cilia move the fertilized egg to the uterus. In addition, epithelial cilia present early in development are involved in left-right axis determination. Some epithelial cells, such as retinal photoreceptor cells and certain renal epithelial cells, possess immotile cilia which we now know play important sensory roles in cell function. Individuals with motility impaired cilia / flagella or defects in ciliary / flagellar assembly may have any number of serious disorders including hydrocephaly, retinal degeneration, respiratory distress, polycystic kidney disease, and infertility.
Despite the diversity of cell types utilizing these organelles for motile functions, the architecture and the molecules which construct ciliary and flagellar axonemes are highly conserved. The motility of these organelles requires the precise regulation of at least four different dynein motor complexes. This regulation is mediated in part by a signal transduction pathway that includes a combination of kinases and phosphatases anchored to the axoneme. Our goal is to understand how these signal transduction pathways are integrated to produce the complex waveforms typical of beating cilia and flagella. Using the biflagellate green alga Chlamydomonas reinhardtii as a model system, we have the most complete and advanced array of biochemical, molecular, and genetic techniques with which to study flagellar motility. The high degree of conservation in axonemal structure and composition across species is well illustrated by the finding that the amino acid sequences of virtually all flagellar proteins in Chlamydomonas share high sequence identity with proteins predicted from the human genome sequence. In fact, several genes in which mutations result in human disease were first identified in Chlamydomonas.
Current projects include:
1. Identifying calmodulin binding proteins in the axoneme.
For virtually
all cell types with cilia and flagella, modulating ciliary and flagellar beating
involves changes in intraflagellar free calcium concentration. Using Chlamydomonas as a model system, we have
identified a role for calmodulin and a calmodulin dependent kinase in a signal
transduction pathway that regulates dynein activity. Using a highly specific
antibody we generated against Chlamydomonas
calmodulin, we have successfully immunoprecipitated at least two different calmodulin
containing complexes and have identified the constituent polypeptides of these
complexes using mass spectrometry. Current projections include biochemically characterizing
these calmodulin binding proteins as well as determining their function using
RNAi technology.
2. Calmodulin dependent kinase (CamKinase).
We have
substantial evidence to indicate that calmodulin dependent kinase is an
axonemal component and plays an important role in regulating motility. There
are several Chlamydomonas ESTs with
substantial homology to known CaM-kinases. Potential projects include
determining which, if any, of these CaM-kinases are flagellar components. We
would also like to identify potential targets of these axonemal CaM-kinases.
These analyses include examining phosphorylation levels of axonemal components
in response to changes in calcium concentration and in the presence of specific
inhibitors.
Recent Publications
Wargo, M. and E.F. Smith (2003)
Asymmetry of the central apparatus defines the location of active microtubule
sliding in Chlamydomonas.
Proceedings of the
Smith, E.F. and P. Yang (2004) The radial spokes and central apparatus: mechano-chemical
sensors for
modulating ciliary and flagellar motility. Cell Motility and the Cytoskeleton 57:8-17.
DiBella, L.M., E.F.
Smith, R. Patel-King, K. Wakabayashi, and S.M. King. (2004) A novel Tctex2-
related light chain is required for stability and motor
activity of inner dynein arm I1 from the
Chlamydomonas flagellum.
Journal of Biological Chemistry
279(20):21666-21676.
Wargo, M., M.A.
McPeek, and E.F. Smith. (2004)
Analysis of microtubule sliding patterns in Chlamydomonas
flagellar axonemes reveals dynein activity on specific doublet
microtubules Journal of Cell Science
117(12):2533-2544.
Dymek, E.E. P.A.
Lefebvre and E.F. Smith. (2004)
PF15 is the Chlamydomonas homologue
of the katanin p80
subunit and is required for assembly of the flagellar central
apparatus. Eukaryotic Cell
3(4):870-879.
Dymek, E.E., M. Wargo, H.P. Benson
and E.F. Smith. Calmodulin is a
component of at least two
different axonemal complexes in Chlamydomonas flagella. (in preparation)