CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2010; 190(5):927-40. DOI: 10.1083/jcb.201006105
Source: PubMed


Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.

    • "Several proteins associated with ciliopathies such as MeckeleGruber syndrome, nephronophthisis and Joubert syndrome localize specifically to the TZ. Here, these proteins regulate the composition and signaling capacity of the organelle by controlling ciliary protein entry and exit, and early steps of ciliogenesis such as centriolar migration and docking with the plasma membrane (Chih et al., 2011; Craige et al., 2010; Dawe et al., 2007; GarciaGonzalo et al., 2011; Huang et al., 2011; Reiter et al., 2012; Williams et al., 2011). Research in the nematode Caenorhabditis elegans has provided important insight into the function of the TZ and associated ciliopathy proteins. "
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    ABSTRACT: The transition zone (TZ) at the ciliary base has emerged as an important regulator of the composition and functions of cilia, which are microtubule-based structures extending from the surfaces of most eukaryotic cells, serving motility, chemo-/mechano-/photosensation and developmental signaling roles. Possessing distinct ultrastructural features such as microtubule-membrane spanning Y-links, the ∼0.2-1.0-μm long TZ is thought to act as a gated cytosolic (size dependent) and membrane diffusion barrier that drives ciliary compartmentalization by preventing unregulated protein exchange between the cilium and the rest of the cell. Multiple proteins associated with ciliary diseases (ciliopathies) such as Meckel-Gruber syndrome (MKS) and nephronophthisis are specifically found in the TZ, and work from a number of model systems, including Chlamydomonas reinharditii, Caenorhabditis elegans and the mouse indicates TZ-gating and associated ciliogenic functions for a number of these proteins. Here we present a suite of assays for probing the structure, function, and molecular composition of the C. elegans TZ, with emphasis on TZ ultrastructure, diffusion barrier kinetics, MKS module assembly hierarchy, and TZ-dependent behaviors. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Dec 2015 · Methods in cell biology
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    • ", whereas no POC genes are in this subset ( Supplemental Data Set 7 ) . Although the specific functions of most POC genes are unknown , the POC3 / CEP290 gene product localizes mainly to the transition zone and is required for flagella assembly , and therefore does not match our prediction for in - volvement in basal body assembly or replication ( Craige et al . , 2010 ) . Three additional large data sets of candidate flagella / basal body protein coding genes were investigated based on their synchronous expression profiles : genes encoding proteins of the flagella pro - teome ( FAPs ) ( Pazour et al . , 2005 ) , CiliaCut genes that are con - served in ciliated species but missing from species without"
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    ABSTRACT: The green alga Chlamydomonas reinhardtii is a useful model organism for investigating diverse biological processes, such as photosynthesis and chloroplast biogenesis, flagella and basal body structure/function, cell growth and division, and many others. We combined a highly synchronous photobioreactor culture system with frequent temporal sampling to characterize genome-wide diurnal gene expression in Chlamydomonas. Over 80% of the measured transcriptome was expressed with strong periodicity, forming 18 major clusters. Genes associated with complex structures and processes, including cell cycle control, flagella and basal bodies, ribosome biogenesis, and energy metabolism, all had distinct signatures of coexpression with strong predictive value for assigning and temporally ordering function. Importantly, the frequent sampling regime allowed us to discern meaningful fine-scale phase differences between and within subgroups of genes and enabled the identification of a transiently expressed cluster of light stress genes. Coexpression was further used both as a data-mining tool to classify and/or validate genes from other data sets related to the cell cycle and to flagella and basal bodies and to assign isoforms of duplicated enzymes to their cognate pathways of central carbon metabolism. Our diurnal coexpression data capture functional relationships established by dozens of prior studies and are a valuable new resource for investigating a variety of biological processes in Chlamydomonas and other eukaryotes.
    Full-text · Article · Oct 2015 · The Plant Cell
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    • "In some experiments, palmelloid cells were induced to grow flagella by treatment with autolysin prepared by the method of Craige et al. (2010). "
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    ABSTRACT: Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation ssh1 (suppressor of shortness 1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in the polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly. © 2015 by The American Society for Cell Biology.
    Preview · Article · Jun 2015 · Molecular biology of the cell
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