Progress 07/01/06 to 12/31/10
Outputs
OUTPUTS: OUTCOMES: Even though funding for this project was extremely limited, progress was made through our exploitation of the model organism, Chlamydomonas reinhardtii. Multiple interactions between the Chlamydomonas IFT particle proteins were identified; some of these interactions were shown to be true for the corresponding mammalian IFT proteins. Interestingly, the complex B protein known as IFT43 was found to interact with multiple B subunits, suggesting that IFT43 acts a scaffolding protein to hold the B complex together. Furthermore, IFT43 was found to significantly increase the solubility of a number of IFT B proteins, suggesting that IFT43 might also exhibit molecular chaperone activities. One of the more valuable Outcomes from the past four years was the discovery that very high centrifuge speeds that generate centrifugal forces of 200,000-300,000 times that of gravity, are capable of forcing the sensitive IFT B complex to fall apart; this is due presumably to the shear forces encountered when moving quickly through the viscous solution. This finding has reshaped our experimental protocols when pursuing the isolation of very large protein complexes. IMPACTS: Found in diverse organisms, cilia and flagella are used to generate movement and often function as sensory organelles. The importance of these functions to human health has become increasingly obvious in just the past few years as cilia and flagella have been linked with no less than 15 human diseases. Known collectively as ciliopathies, these diseases include the most common life-threatening genetic disease, polycystic kidney disease (PKD; affects ~1/500 adults), as well as Bardet-Beidl Syndrome (BBS), Primary Cilia Dyskinesia (PCD), Kartagener's Syndrome, Meckel-Gruber Syndrome, hydrocephalus, and retinal degeneration. We are especially proud that our 2000 publication (Pazour et al, J. Cell Biol.; cited ~350 times in the past decade) characterizing the IFT protein IFT88/Tg737 was the first to indicate that cilia and IFT were associated with PKD; research from many outstanding laboratories since then have proven our original finding to be correct. Cilia and flagella have also been found to mediate specific signaling pathways that are especially important to the proper development of embryos, fetuses and young children. These pathways include the sonic hedgehog (Shh) and wingless (Wnt) signaling pathways; IFT is directly involved as it has been shown to be required for the delivery of key signaling pathway components within the organelles. We will continue, likely in collaboration with medical researchers, to explore the association between IFT and human and animal disease and development as we continue our molecular characterization of this intracellular transport process. PARTICIPANTS: Douglas G Cole, Principal Investigator (10%). Responsible for overall strategic planning and execution of specific experiments. Mark S Miller, Scientific Aide/Technician (78%). Responsible for experimental execution and laboratory stocks maintenance. TARGET AUDIENCES: Our target audience is the biomedical research community with particular emphasis on those interested in the assembly and function of cilia and flagella and understanding the role these organelles play in human health. In the past four years, our results were delivered to this audience through multiple primary research and review articles in biomedical research journals that include the Journal of Biological Chemistry and Cell. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
ACTIVITIES: Intraflagellar transport (IFT) is an ancient and conserved transport mechanism required for the assembly, maintenance and function of all eukaryotic cilia and flagella. Best characterized in the biflagellate unicellular green algae, Chlamydomonas, the proteinaceous IFT particles are moved along the length of the organelle by the microtubule-based motor proteins, kinesin-2 and cytoplasmic dynein 1b/2. Most of our experimental research in the past four years has focused on the structure and function of the protein complexes, A and B, which form the IFT particles. These studies have included analysis of protein-protein interactions and the identification and characterization of mutant algal strains that are defective in A or B genes. Lastly, we identified a putative cargo protein that interacts directly with the retrograde IFT motor known as dynein 1b. EVENTS & DISSEMINATION: In addition to numerous publications, our findings were disseminated in the past four years at a variety of regional, national and international scientific meetings and through invited research seminars delivered at Universities on three continents. Meeting highlights include invitations to speak at two FASEB Meetings (2007, 2010), two Gordon Conferences (2006, 2007), and an international EMBO Workshop in the South of France (2008). Highlights of invited seminars include the Marie Curie Sklowdoska University in Lublin, Poland (2008), the University of California, Davis (2009), and Tsinghua University in Beijing, China (2010). SERVICES: Two talks were given to general audiences. The first talk was entitled "Unveiling the Secrets: Pond Scum and Human Disease" and was presented to a general lay audience in the Health Seminar Series on the evening of April 15 (2008) at the North Idaho College in Coeur d'Alene, ID). A second talk entitled "Why Does the NIH Fund Research on Pond Scum" was presented to a broad undergraduate audience as part of the INBRE Summer Seminar Series on June 24 (2009) at the University of Idaho in Moscow, ID). Both talks focused on how green algal research has enhanced discoveries in human ciliopathy diseases such as polycystic kidney disease. PRODUCTS: The laboratory graduated one Masters student, Micah Ferrell; his thesis was entitled "Interaction Analysis of Intraflagellar Transport Proteins." Experimental highlights include the development of an assay to electroporate purified recombinant protein (IFT46) into a mutant cell lines (unable to produce IFT46) and were able to rescue the cells' ability to assemble flagella and swim. We have also developed a new procedure to isolate very large protein complexes. We believe our approach could be adopted by others to identify novel complexes isolated from other organisms.
Publications
- Fan FZ, Behal RH, Geimer S, Wang Z, Williamson SM, Zhang H, Cole DG, and Qin H. (2010) Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly. Mol. Biol. Cell, 21:2696-2706. PMCID: PMC2912355
- Lucker BF, Miller MS, Dziedzic SA, Blackmarr PT, and Cole DG. (2010) Direct interactions of intraflagellar transport complex B proteins IFT88, IFT52 and IFT46. J. Biol. Chem. 285:21508-21518. PMCID: PMC2898376
- Betleja E, and Cole DG. (2010) Ciliary trafficking: CEP290 guards a gated community. Curr. Biol. 20:R928-931. PMID: 21056833
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: ACTIVITIES: Intraflagellar transport (IFT) is an ancient and conserved transport mechanism required for the assembly, maintenance and function of all eukaryotic cilia and flagella. Best characterized in the biflagellate unicellular green algae, Chlamydomonas, the proteinaceous IFT particles are moved along the length of the organelle by the microtubule-based motor proteins, kinesin-2 and cytoplasmic dynein 1b/2. Most of our experimental research in the past year has focused on our continuing analysis of the structure and function of the protein complexes, A and B, which form the IFT particles. These studies include analysis of protein-protein interactions and the continued search for mutant strains that are defective in A or B genes. Lastly, we have continued to pursue the identification of cargo proteins that interact directly with the IFT motor proteins. EVENTS & DISSEMINATION: In addition to three publications, our findings were disseminated in the past year at a research seminar given to the Department of Molecular and Cellular Biology at the University of California, Davis (10/01/2009) and at the 49th Annual Meeting of the American Society for Cell Biology, San Diego, CA (12/07/2009). SERVICES: A talk entitled "Why Does the NIH Fund Research on Pond Scum" was presented to a broad undergraduate audience as part of the INBRE Summer Seminar Series on June 24 (2009) at the University of Idaho in Moscow, ID). This talk focused on how green algal research has enhanced discoveries in human ciliopathy diseases such as polycystic kidney disease. PRODUCTS: We have developed a new procedure to isolate very large protein complexes. We believe our approach could be adopted by others to identify novel complexes isolated from other organisms. PARTICIPANTS: Douglas G Cole, Principal Investigator (10%). Responsible for overall strategic planning and execution of specific experiments. Mark S Miller, Scientific Aide/Technician (78%). Responsible for experimental execution and laboratory stocks maintenance. TARGET AUDIENCES: Our target audience is the biomedical research community with particular emphasis on those interested in the assembly and function of cilia and flagella and understanding the role these organelles play in human health. In 2009, our results were delivered to this audience through one review publication in the Cell (Cole & Snell, 2009), one review chapter in "The Chlamydomonas Sourcebook," one protocols chapter in "Methods in Cell Biology, Volume 92." PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES: (1) Interactions between IFT complex A subunits were further characterized. As described previously, bacterially expressed IFT43 interacts well with IFT121. We now have evidence that IFT43 may also interact with each of the other complex A subunit proteins. We have been unable to identify direct interactions between any of the other complex A subunits. If we can verify the IFT43 interactions, it will suggest that IFT43 acts as a `protein glue' to hold the complex together. Such a hypothesis will be tested by generation of an IFT43 mutant strain or through knockdown of IFT43 gene expression. (2) Analysis of the IFT complex B has been advanced by the co-expression and co-purification of sub-complex containing five different B subunit proteins. In vitro assembly of higher order complexes is an ongoing project. (3) Analysis of flagellar extracts has long benefited from spreading the proteins out by centrifugation through a sucrose density gradient. We discovered this past year, however, that increased centrifuge speeds can cause sensitive protein complexes such as IFT B, to fall apart due presumably to shear forces encountered when moving through the viscous solution. This finding has us rethinking our purification methods. IMPACTS: Found in diverse organisms, cilia and flagella are used to generate movement and often function as sensory organelles. The importance of these functions to human health has become increasingly obvious in just the past few years as cilia and flagella have been linked with a number of human diseases. Known collectively as ciliopathies, these diseases include the most common life-threatening genetic disease, polycystic kidney disease (PKD; affects ~1/500 adults), as well as Bardet-Beidl Syndrome (BBS), Primary Cilia Dyskinesia (PCD), Kartagener's Syndrome, Meckel-Gruber Syndrome, hydrocephalus, and retinal degeneration. We are especially proud that our 2000 publication (Pazour et al, J. Cell Biol.; cited over 300 times in the past decade) characterizing the IFT protein IFT88/Tg737 was the first to indicate that cilia and IFT were associated with PKD; research from many outstanding laboratories since then have proven our original finding to be correct. Cilia and flagella have also been found to mediate specific signaling pathways that are especially important to the proper development of embryos, fetuses and young children. These pathways include the sonic hedgehog (Shh) and wingless (Wnt) signaling pathways; IFT is directly involved as it has been shown to be required for the delivery of key signaling pathway components within the organelles. We will continue, likely in collaboration with medical researchers, to explore the association between IFT and human and animal disease and development as we continue our molecular characterization of this intracellular transport process.
Publications
- Cole DG, Snell WJ. (2009) Snapshot: Intraflagellar transport. Cell 15:784-784.e1.
- Behal, RH, Betleja, E, and Cole, DG. (2009) Purification of IFT Particle Proteins and Preparation of Recombinant Proteins for Structural and Functional Analysis. In "Methods in Cell Biology, Volume 92, Cilia: Motors and Regulation, Chapter 10 (pg 179-196)" (S. King and G. Pazour, Ed.) Academic Press, San Diego.
- Cole, DG (2009). Intraflagellar Transport. In "The Chlamydomonas Sourcebook: Cell Motility and Behavior, 2nd Edition, Volume 3, Chapter 4 (pg 71-112)." (G. Witman and E. Harris, Ed.) Academic Press, San Diego.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: ACTIVITIES: Intraflagellar transport (IFT) is an ancient and conserved transport mechanism required for the assembly, maintenance and function of all eukaryotic cilia and flagella. Best characterized in the biflagellate unicellular green algae, Chlamydomonas, the proteinaceous IFT particles are moved along the length of the organelle by the microtubule-based motor proteins, kinesin-2 and cytoplasmic dynein 1b/2. Most of our experimental research in the past year has focused on our continuing analysis of the structure and function of the protein complexes, A and B, which form the IFT particles. Since the nature of complex A is poorly understood, we have focused much of our efforts on the architectural analysis of complex A. These studies include analysis of protein-protein interactions and the continued search for mutant strains that are defective in complex A genes. We have also continued on our studies of the complex B subunit known as IFT46 through collaborations with Dave Mitchell (SUNY Upstate Medical University). Lastly, we have pursued identification of cargo proteins that interact directly with the IFT motor proteins. EVENTS & DISSEMINATION: In addition to publications, our findings were disseminated in the past year at several 2008 scientific meetings including (1) the 13th International Conference and 1st EMBO Workshop on the Cell and Molecular Biology of Chlamydomonas, Hyeres-les-Palmiers, France, May 27-June 01; (2) the Center for Reproductive Biology Annual Retreat in Orofino, ID, May 15-16; and (3) the 48th Annual Meeting of the American Society for Cell Biology, San Francisco, CA, December 13-17. SERVICES: A talk entitled "Unveiling the Secrets: Pond Scum and Human Disease" was presented to a general lay audience in the Health Seminar Series on the evening of April 15 (2008) at the North Idaho College in Coeur d'Alene, ID). In addition to describing the how green algal research has enhanced discoveries in human ciliopathy diseases such as polycystic kidney disease, the talk also illustrated emerging agricultural uses for algae including food sources and biofuel generation. PRODUCTS: The laboratory graduated one Masters student, Micah Ferrell; his thesis was entitled "Interaction Analysis of Intraflagellar Transport Proteins." We also developed an assay where we electroporate purified IFT46 protein into a mutant cell line (unable to produce IFT46) and are able to rescue ~25% of the cells so that after 2-4 hours, the cells assemble flagella and are able to swim. This technique is now being adopted by other Chlamydomonas researchers. PARTICIPANTS: Douglas G Cole, Principal Investigator (10%). Responsible for overall strategic planning and execution of specific experiments. Mark S Miller, Scientific Aide/Technician (78%). Responsible for experimental execution and laboratory stocks maintenance. TARGET AUDIENCES: Our target audience is the biomedical research community with particular emphasis on those interested in the assembly and function of cilia and flagella and understanding the role these organelles play in human health. In 2008, our results were delivered to this audience through one research publication in the Journal of Cell Biology (Ahmed et al., 2008), one review chapter in "The Chlamydomonas Sourcebook," and through presentations and discussions at three regional, national and international meetings. Regional Meetings: Annual Retreat, Center for Reproductive Biology, Orofino, ID. National Meeting: Annual Meeting of the American Society for Cell Biology, San Francisco, CA. International Meeting: EMBO Workshop on the Cell and Molecular Biology of Chlamydomonas, Hyeres-les-Palmiers, France. We have also targeted a general audience by delivering a Health Series Seminar (Coeur d'Alene, ID) to general laypeople regarding the role that basic research using model organisms plays in providing key information in the advancement of understanding human and animal disease. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
OUTCOMES: (1) In a recently published collaborative effort, the complex B subunit, IFT46 was shown to interact with ODA16, a protein required for effective transport (presumably via IFT) of outer dynein arms into Chlamydomonas flagella (Ahmed et al., 2008, J. Cell Biol. 183:313). The putative interaction between IFT46 and ODA16 was confirmed through pulldowns of recombinant protein as well as co-immunoprecipitations of native proteins from flagellar extracts. (2) We have expanded on an earlier finding that we could effectively rescue ift46 mutants through in vivo electroporation of purified IFT46 protein. The protein is expressed in E coli as a fusion protein with a histidine tag (six histidine residues). The purified protein was then chemically labeled with a fluorescent marker (Alexafluor 488) prior to electroporation. Cells that were effectively rescued were able to assemble full length flagella within four hours. Fluorescent imaging allowed us to visualize the movement of the labeled-IFT particles along the length of the organelle. We hope to exploit this assay in the future by combining two or more fluorescent labels to study the dynamics of IFT. (3) Interactions between complex A subunits, IFT121 and IFT43 (formerly IFT42), were further characterized. Deletion analysis showed that the carboxyl-terminal half of IFT121 is necessary and sufficient for interaction with IFT43. Further interaction analysis is awaiting better characterization of putative IFT121 and IFT43 mutant strains. IMPACTS: Found in diverse organisms, cilia and flagella are used to generate movement and often function as sensory organelles. The importance of these functions to human health has become increasingly obvious in just the past few years as cilia and flagella have been linked with a number of human diseases. Known collectively as ciliopathies, these diseases include the most common life-threatening genetic disease, polycystic kidney disease (PKD; affects ~1/500 adults), as well as Bardet-Beidl Syndrome (BBS), Primary Cilia Dyskinesia (PCD), Kartagener's Syndrome, Meckel-Gruber Syndrome, hydrocephalus, and retinal degeneration. We are especially proud that our 2000 publication (Pazour et al, J. Cell Biol.) characterizing the IFT protein IFT88/Tg737 was the first to indicate that cilia and IFT were associated with PKD; research from many outstanding laboratories since then have proven our original finding to be correct. Cilia and flagella have also been found to mediate specific signaling pathways that are especially important to the proper development of embryos, fetuses and young children. These pathways include the sonic hedgehog (Shh) and wingless (Wnt) signaling pathways; IFT is directly involved as it has been shown to be required for the delivery of key signaling pathway components within the organelles. We will continue, likely in collaboration with medical researchers, to explore the association between IFT and human and animal disease and development as we continue our characterization of this intracellular transport process.
Publications
- Cole, DG (2008). Intraflagellar Transport. (G. Witman and E. Harris, Ed.)Chapter 4 in The Chlamydomonas Sourcebook: Cell Motility and Behavior, 2nd Edition, Volume 3 (pg 71-112). Academic Press, San Diego.
- Redding KE, Cole DG. (2008) Chlamydomonas: a sexually active, light-harvesting, carbon-reducing, hydrogen-belching 'planimal'. EMBO Rep. 9:1182-1187.
- Ahmed NT, Gao C, Lucker BF, Cole DG, Mitchell DR. (2008) ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. J Cell Biol. 183:313-322.
- Behal RH, Jones A, Miller MS, Rea C, Cole DG. (2008) Interaction Analysis of IFT Complex A Subunits IFT43 and IFT121. 48th Annual Meeting of the American Society for Cell Biology (Abstracts CD-ROM), San Francisco, CA.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Intraflagellar transport (IFT) is an ancient and conserved transport mechanism required for the assembly, maintenance and function of all eukaryotic cilia and flagella. Best characterized in the biflagellate unicellular green algae, Chlamydomonas, the proteinaceous IFT particles are moved along the length of the organelle by the microtubule-based motor proteins, kinesin-2 and cytoplasmic dynein 1b/2. Most of our progress in the past year has resulted from our analysis of the structure and function of the protein complexes, A and B, which form the IFT particles. Since the nature of complex A is poorly understood, we have now focused on an architectural analysis of complex A. Using yeast-based two-hybrid analysis, we looked for interactions among the complex A subunits and have identified a direct interaction between the complex A proteins, IFT121 and IFT42. Further characterization of this interaction is currently underway. Our most interesting development in the study of IFT
complex B is that the core subunit, IFT46, is capable of interacting with multiple protein domains. Previously we found that IFT46 interacts strongly with the neighboring complex B subunits, IFT52 and IFT88. Here we report that IFT46 interacts equally well with the amino- and carboxy-terminal halves of IFT88. Further deletion analysis has revealed that IFT46 interacts with two separate and distinct TPR domains found within IFT88. Since these results suggested that IFT46 might have a general affinity for TPR domains, we tested additional TPR-containing IFT subunits and found that IFT46 was able to bind to both the N- and C-terminal halves of the complex A TPR protein, IFT139, and with the putative IFT protein, DYF-1. In order to test a more divergent TPR protein, we co-expressed the MBP-IFT46F with STI-1, a yeast co-chaperone protein that contains three TPR domains (TPR1, TPR2A, TPR2B) arranged similarly to the IFT88 (Flom et al., 2007, Biochem. J. 404:159). The Chlamydomonas IFT46
failed to interact with the yeast STI-1. Future experiments will address the specificity of IFT46 interaction with multiple proteins. Lastly, one of the functions of IFT is to move protein cargo along the flagella, for assembly and disassembly of the flagellum, and for other purposes such as mediation of inter-cellular interactions. In an attempt to identify specific IFT cargos, we have employed a yeast-based two-hybrid library screen using a pool of the complex A subunits. The initial screens have identified several putative cargos that are currently undergoing further analysis. Our findings were disseminated in the past year at several 2007 scientific meetings including invited talks at (1) the Gordon Research Conference on Cilia, Mucus and Mucociliary Interactions in Ventura, Ca; (2) the Center for Reproductive Biology Annual Retreat in Orofino, ID; (3) the FASEB Summer Research Conference on the Biology of Cilia and Flagella in Saxtons River, VT; and a poster at the Northwest
Reproductive Sciences Symposium in Moscow, ID.
PARTICIPANTS: Douglas G Cole, Prinicipal Investigator (10%). Responsible for overall strategic planning and execution of specific experiments. Mark S Miller, Scientific Aide/Technician (80%). Responsible for experimental execution and laboratory stocks maintenance.
TARGET AUDIENCES: Our target audience is the biomedical research community with particular emphasis on those interested in the assembly and function of cilia and flagella and understanding the role these organelles play in human health. In 2007, our results were delivered to this audience through one publication in the Biochemical Journal (Flom et al., 2007) and through presentations at four regional, national and international meetings. Regional Meetings: Poster at the Northwest Reproductive Sciences Symposium, Moscow, ID, March 23-24, 2007; Invited oral presentation at 11th Annual Retreat, Center for Reproductive Biology, Orofino, ID, May 24-25, 2007. National Meeting: Invited oral presentation at Cilia, Mucus & Mucociliary Interactions Gordon Research Conference, Ventura, CA, February 4-9, 2007. International Meeting: Invited oral presentation at the FASEB Summer Research Conference on the Biology of Cilia and Flagella, Saxtons River, VT, August 4-9, 2007.
PROJECT MODIFICATIONS: We have no major changes to declare at this time.
Impacts
Found in diverse organisms, cilia and flagella are used to generate movement and often function as sensory organelles. The importance of these functions to human health has become increasingly obvious in just the past few years as cilia and flagella have been linked with a number of human diseases. Known collectively as ciliopathies, these diseases include the most common life-threatening genetic disease, polycystic kidney disease (PKD; affects ~1/500 adults), as well as Bardet-Beidl Syndrome (BBS), Primary Cilia Dyskinesia (PCD), Kartagener's Syndrome, Meckel-Gruber Syndrome, hydrocephalus, and retinal degeneration. We are especially proud that our 2000 publication (Pazour et al, J. Cell Biol.) characterizing the IFT protein IFT88/Tg737 was the first to indicate that cilia and IFT were associated with PKD; research from many outstanding laboratories since then have proven our original finding to be correct. Cilia and flagella have also been found to mediate specific
signaling pathways that are especially important to the proper development of embryos, fetuses and young children. These pathways include the sonic hedgehog (Shh) and wingless (Wnt) signaling pathways; IFT is directly involved as it has been shown to be required for the delivery of key signaling pathway components within the organelles. We will continue, likely in collaboration with medical researchers, to explore the association between IFT and human and animal disease and development as we continue our characterization of this intracellular transport process.
Publications
- Flom, G, Behal RH, Rosen L, Cole DG, Johnson JL. (2007) Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions. Biochem. J., 404:159-167.
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Progress 01/01/06 to 12/31/06
Outputs
Intraflagellar transport (IFT) is an ancient and conserved transport mechanism required for the assembly, maintenance and function of all eukaryotic cilia and flagella. Best characterized in the biflagellate unicellular green algae, Chlamydomonas, the proteinaceous IFT particles are moved along the length of the organelle by the microtubule-based motor proteins, kinesin-2 and cytoplasmic dynein 1b/2. Most of our progress in the past year has come through the development of important tools, including mutant strains, antibodies and a new biological assay, which we will use to dissect structures and functions of IFT components. Using insertional mutagenesis, we have identified two new IFT mutants; these strains are disrupted in the IFT121 and FLA10 genes, respectively. Both of these mutant cell lines have extremely short flagellar stubs indicating that both genes are required for flagellar assembly; the FLA10 result confirms earlier studies while the IFT121 phenotype is
the first mutant in this gene in any ciliated organism. We have also succeeded in generating specific antisera against the IFT42 subunit. Essentially nothing is known about this gene and its protein product other than it is a component of IFT complex A. We will use this new antisera to address cell biological issues such as temporal and spatial subcellular localizations and to address biochemical issues such as associations with other IFT proteins. Lastly, we have developed a biological assay for screening the biological activity of IFT proteins that have been expressed and purified from bacteria. In brief, the assay uses a strong pulse of an electric field to force the purified proteins to enter cells. We have used this protein electroporation technique to replace, temporarily, IFT46 protein into a mutant cell line that lacks the IFT46 gene. Within 2 hours, IFT46 mutant cells that receive purified intact IFT46 protein regain full-length flagella and are able to swim in a normal
fashion; mutant cells that receive a control protein remain flagella-less and are unable to swim. Using this approach we have found that the first 25 amino acids of the IFT46 protein are not required for flagellar assembly; removal of 50 or more amino acids, however, generates a protein that is unable to rescue flagellar assembly. We plan to use the protein electroporation to further characterize functional domains with IFT46 and additional IFT proteins which can be successfully reintroduced into the appropriate mutant cells.
Impacts
Found in diverse organisms, cilia and flagella are used to generate movement and often function as sensory organelles. The importance of these functions to human health has become increasingly obvious in just the past few years as cilia and flagella have been linked with a number of human diseases. Known collectively as ciliopathies, these diseases include the most common life-threatening genetic disease, polycystic kidney disease (PKD; affects ~1/500 adults), as well as Bardet-Beidl Syndrome (BBS), Primary Cilia Dyskinesia (PCD), Kartagener's Syndrome, Meckel-Gruber Syndrome, hydrocephalus, and retinal degeneration. We are especially proud that our 2000 publication (Pazour et al, J. Cell Biol.) characterizing the IFT protein IFT88/Tg737 was the first to indicate that cilia and IFT were associated with PKD; research from many outstanding laboratories since then have proven our original finding to be correct. Cilia and flagella have also been found to mediate specific
signaling pathways that are especially important to the proper development of embryos, fetuses and young children. These pathways include the sonic hedgehog (Shh) and wingless (Wnt) signaling pathways; IFT is directly involved as it has been shown to be required for the delivery of key signaling pathway components within the organelles. We will continue, likely in collaboration with medical researchers, to explore the association between IFT and human and animal disease and development as we continue our characterization of this intracellular transport process.
Publications
- No publications reported this period
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