Publications (72) View all
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Article: Analysis of Ciliary Membrane Protein Dynamics Using SNAP Technology.
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ABSTRACT: The sensory functions of the primary cilium rely on receptors and other membrane proteins that are specifically sorted to the ciliary compartment, which is a subdomain of the plasma membrane. Defects in this process underlie a large number of human diseases, yet it is poorly understood. Thus, it is of great interest to understand the mechanisms by which the cell sorts and traffics proteins to the ciliary membrane. Here, we provide an overview of our method to study the sorting and trafficking of ciliary membrane proteins using SNAP technology. This technology enables pulse-chase analysis of the movement of proteins through the endomembrane system and onto the cilium.Methods in enzymology 01/2013; 524:195-204. · 1.90 Impact Factor -
Article: The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function.
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ABSTRACT: The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.The Journal of Cell Biology 10/2012; 199(1):151-67. · 10.26 Impact Factor -
Article: IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport.
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ABSTRACT: The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.Developmental cell 05/2012; 22(5):940-51. · 13.36 Impact Factor -
Article: Disruption of IFT complex A causes cystic kidneys without mitotic spindle misorientation.
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ABSTRACT: Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney-specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre-driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pronounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression.Journal of the American Society of Nephrology 01/2012; 23(4):641-51. · 9.66 Impact Factor -
Article: A unified taxonomy for ciliary dyneins.
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ABSTRACT: The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.Cytoskeleton 09/2011; 68(10):555-65.
