RESEARCH INTERESTS
Assembly and function of eukaryotic cilia and flagella
BACKGROUND
Cilia and flagella are highly dynamic microtubule (MT)-based organelles that assemble from and continuously turn over at the flagellar tip. These organelles emanate from the surface of many eukaryotic cells and have diverse roles in motility and sensory perception. Motile cilia/flagella enable the cell (e.g. a sperm cell) to propel itself in the right direction or function in the transport of fluids over ciliated surface epithelia, for example in the airways. Both motile and non-motile (primary) cilia display specific receptors and ion channels and function as sensors involved in regulating tissue homeostasis and cell growth. Proper assembly and maintenance of cilia and flagella are therefore critical to human health, and absent or defective cilia/flagella have been associated with severe human diseases such as respiratory disease, polycystic kidney disease, retinal degenerative disease, infertility, defective left-right-axis determination, and Bardet-Biedl syndrome.
RESEARCH PROJECTS
1) Regulation of intraflagellar transport at the flagellar tip
The assembly and maintenance of cilia/flagella depend on a process called intraflagellar transport (IFT). IFT is a bidirectional MT-based motility system that transports flagellar precursors from the cell body to the flagellar tip for assembly and returns flagellar turnover products from the tip to the cell body. The IFT system consists of anterograde (base to tip) and retrograde (tip to base) MT motor complexes associated with groups of large protein complexes called IFT particles (Figure 1). Genetic and biochemical analyses in model organisms such as the flagellated green alga Chlamydomonas have led to the identification of most of the IFT motor subunits as well as most of the IFT particle polypeptide genes. These studies have been instrumental for understanding the molecular basis of ciliary diseases like polycystic kidney disease and retinal degeneration. Despite the physiological importance of IFT, many aspects of this process remain poorly understood. For example, the molecular mechanisms by which different components of the IFT system interact and are regulated are unknown.
This project focuses on the molecular mechanisms of IFT turnaround at the flagellar tip. Using Chlamydomonas as a model system, we recently proposed a model for IFT in which tip turn-around consists of three distinct steps (Pedersen et al., 2006, Curr Biol 16: 450-459). We are currently using biochemical, molecular, and genetic approaches to test this model and to investigate how each of the three steps is regulated.
Figure 1. Model for IFT in Chlamydomonas. Step 1: gathering of IFT particles and motors in the peri-basal body region. Step 2: kinesin-2-mediated anterograde transport of IFT complexes A and B and inactive cDynein1b. Step 3: release of complexes A and B and inactive cDynein1b into the flagellar tip compartment, followed by their dissociation from each other. Step 4: complex A binds to active cDynein1b via the LIC subunit, complex B then re-associates with complex A, and kinesin-2 binds to active cDynein1b independent of complexes A and B and cDynein1b LIC. Step 5: active cDynein1b transports everything back to the cell body. Step 6: IFT components are recycled to the cell body. For details see Pedersen et al. 2006, Curr Biol 16: 450-459.
2) Identification and characterization of flagellar tip proteins
Some of the proteins involved in regulating IFT at the flagellar tip are likely to be localized specifically to this site. We previously showed that the small MT plus end-tracking protein EB1 localizes to the flagellar tip and basal bodies/centrioles in Chlamydomonas (Pedersen et al., 2003, Curr Biol 11: 1969-1974) and interacts with IFT protein 172 (Pedersen et al. 2005, Curr Biol 15: 262-266). We are currently attempting to identify additional binding partners of EB1 in the flagellum, and to study the function of EB1 in the flagellum using RNA interference technology. To this end, we are using both Chlamydomonas and mammalian tissue culture cells as model systems.
Figure 2. Immunofluorescence micrograph of a Chlamydomonas cell showing localization of EB1 (red) to the tip of flagella. The flagella are stained with an antibody against acetylated alpha tubulin (green). From Pedersen et al., 2003, Curr Biol 11: 1969-1974.
3) Localization and function of Lis1 in cilia and flagella
Lissencephaly is a severe developmental brain disorder characterized by a smooth cerebral surface, thickened cortex, and misplaced neurons. Type I lissencephaly is caused by mutations in the LIS1 gene, which encodes a WD-repeat protein previously implicated in cytoplasmic dynein regulation, nuclear migration, and mitosis. Several proteins involved in nuclear migration in Aspergillus bind directly to Lis1, including the nuclear movement protein NudC. Mammalian NudC is highly expressed in ciliated epithelia, and localizes to motile cilia in various tissues, including brain. Moreover, a NudC ortholog is upregulated upon deflagellation in Chlamydomonas, suggesting that it is a flagellar protein. However, the function of NudC in flagella is unknown.
We have cloned a gene encoding a Lis1-like protein (CrLis1) from Chlamydomonas and found that this protein is present in flagella, and interacts with outer dynein arm components. Furthermore, we have found that CrLis1 binds directly to rat NudC indicating that it is a functional ortholog of the mammalian Lis1 protein. These results suggest that Lis1 and NudC are present in cilia/flagella and may play a role in regulating outer arm dynein activity. We are presently investigating whether mammalian Lis1 localizes to motile cilia in the brain and other tissues, and whether Chlamydomonas NudC, like CrLis1, is associated with outer arm dynein components in the flagella.
COLLABORATORS
Søren Tvorup Christensen, Ian H. Lambert, and Gert Christoffersen, Institute of Molecular Biology and Physiology, University of Copenhagen, Denmark
Joel L. Rosenbaum, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
Stefan Geimer, University of Bayreuth, Bayreuth, Germany
Stephen M. King, Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA
CV
Education
PhD in Medical Microbiology, 1996, University of Aarhus, Denmark
MS in Chemistry and Biotechnology, 1994, University of Aarhus, Denmark
Employment
July 15 2005-present: Assistant Professor, Institute of Molecular Biology and Physiology, University of Copenhagen, Denmark
Jan. 2004-July 14 2005: Associate Research Scientist, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
2000-Jan. 2004: Postdoctoral Research Associate, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
Dec. 1999-Mar. 2000: Maternity leave
1997-1999: Postdoctoral Research Fellow, Department of Biochemistry, University of Connecticut Health Center, Farmington, CT, USA
1993-1996: Graduate Student, Department of Medical Microbiology and Immunology, University of Aarhus, Denmark
Aug.-Dec. 1995: Short Term Exchange Student, Department of Biochemistry, University of Connecticut Health Center, Farmington, CT, USA
PUBLICATIONS (FROM 2000-PRESENT)
Pedersen, L. B., Ragkousi, K., Cammett, T. J., Melly, E., Schopick, E., Murray, T., and Setlow, P. 2000. Characterization of ywhE, which encodes a new, putative high-molecular-weight class A penicillin-binding protein in Bacillus subtilis. Gene 246:187-196.
Pedersen, L. B., and Setlow, P. 2000. Penicillin-binding protein-related factor A (PrfA) is required for proper chromosome segregation in Bacillus subtilis. J. Bacteriol. 182: 1650-1658.
Pearson, C.L., Loshon, C.A., Pedersen, L.B., Setlow, B., and Setlow, P. 2000. Analysis of the function of a putative 2,3-diphosphoglyceric acid-dependent phosphoglycerate mutase from Bacillus subtilis. J. Bacteriol. 182:4121-4123.
Pedersen, L. B., Geimer, S., Sloboda, R., and Rosenbaum, J. 2003. The microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii. Curr. Biol. 11: 1969-1974 (+cover).
Pedersen, L. B., Miller, M. S., Geimer, S., Leitch, J. M., Rosenbaum, J. L., and Cole, D. G. 2005. Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip. Curr. Biol. 15: 262-266.
Mitchell, B. F., Pedersen, L. B., Feely, M., Rosenbaum, J. L., and Mitchell, D. R. 2005. ATP production in Chlamydomonas reinhardtii flagella by glycolytic enzymes. Mol. Biol. Cell 16: 4509-4518.
FUNDING
The Danish Natural Science Research Council (rammebevilling)
The Novo Nordisk Foundation
The Carlsberg Foundation (co-applicant with Søren Tvorup Christensen)
MASTERS/BACHELOR PROJECTS (LINK TO 4)