The Rockefeller University Press $30.00
J. Cell Biol. Vol. 190 No. 5 927–940
Correspondence to George B. Witman: George.Witman@umassmed.edu
Abbreviations used in this paper: BAC, bacterial artificial chromosome; BBS,
Bardet-Biedl syndrome; DIC, differential interference contrast; IFT, intraflagellar
transport; LCA, Leber congenital amaurosis.
Mutations that disrupt the assembly, structure, and/or function of
cilia or flagella result in cilia-related disorders termed ciliopathies
(Badano et al., 2006; Fliegauf et al., 2007). Mutations in CEP290
cause ciliopathies that exhibit a range of severity (Helou et al.,
2007). CEP290 mutations are a common cause of Leber congeni-
tal amaurosis (LCA; den Hollander et al., 2006; Sundaresan et al.,
2009), in which blindness results from degeneration of the retina
but other organ systems are often unaffected. Other CEP290 mu-
tations cause Meckel syndrome (Baala et al., 2007; Frank et al.,
2008), a perinatal-lethal disease in which essentially all tissues
with cilia are affected. Of intermediate severity is Joubert syn-
drome, in which brain development is affected and which
often also presents with ocular and renal manifestations (Sayer
et al., 2006; Valente et al., 2006; Brancati et al., 2007).
The described subcellular localization of CEP290 (also
known as nephrocystin-6/NPHP6) is consistent with a role for
CEP290 in cilia. In mammalian cells, CEP290 has been localized
to centrosomes/basal bodies (Andersen et al., 2003; Chang et al.,
2006; Sayer et al., 2006; Valente et al., 2006; Tsang et al., 2008),
pericentriolar satellites (Kim et al., 2008), the connecting cilium
of photoreceptors (Chang et al., 2006; Sayer et al., 2006), and the
dendritic knobs of olfactory sensory neurons (McEwen et al.,
2007), and CEP290 was immunoprecipitated with centrosomal
components (Chang et al., 2006; McEwen et al., 2007; Tsang
et al., 2008). CEP290 has a nuclear localization signal and, in ad-
dition to its cytoplasmic localization, has been observed in the
nucleus (Guo et al., 2004; Sayer et al., 2006).
The exact role of CEP290 is unclear, but all available evi-
dence suggests that CEP290 is somehow involved in ciliary
function. Morpholino knockdown of CEP290 in zebrafish re-
sulted in retinal, renal, and cerebellar phenotypes that are indic-
ative of defects in cilia and are commonly seen in humans with
Joubert syndrome (Sayer et al., 2006). Defects in the localization
of several ciliary proteins were found in photoreceptors and
a Chlamydomonas reinhardtii mutant in which most of the
CEP290 gene is deleted. Immunoelectron microscopy in-
dicated 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
utations in human CEP290 cause cilia-related
disorders that range in severity from isolated
blindness to perinatal lethality. Here, we describe
of isolated flagella revealed that the mutant flagella have
abnormal protein content, including abnormal levels
of intraflagellar transport proteins and proteins asso-
ciated with ciliopathies. Experiments with dikaryons showed
that CEP290 at the transition zone is dynamic and under-
goes 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
CEP290 tethers flagellar transition zone
microtubules to the membrane and regulates
flagellar protein content
Branch Craige,1 Che-Chia Tsao,2 Dennis R. Diener,2 Yuqing Hou,1 Karl-Ferdinand Lechtreck,1 Joel L. Rosenbaum,2
and George B. Witman1
1Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655
2Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520
© 2010 Craige et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub-
lication date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 190 • NUMBER 5 • 2010 928
The Y168 mutant was backcrossed twice to wild-type cells
(the F2 progeny with the deletion is henceforth referred to as
cep290), and each time the mutant phenotype cosegregated with
the deletion. The mutant phenotype was rescued by transforma-
tion with genomic DNA encoding either untagged or HA-tagged
CEP290 and no other gene (Fig. 1, A and B; and Videos 1–4),
which confirmed that the mutant phenotype is caused by the dele-
tion of CEP290. Antibodies generated against a peptide consist-
ing of the last 14 amino acids in the C terminus of C. reinhardtii
CEP290 recognized a single band of the predicted molecular mass
(275 kD) that was present in Western blots of whole-cell lysates
of wild-type and rescued cells, but absent in cep290 cells, which
confirmed that the antibody is specific and that CEP290 is absent
in the mutant (Fig. 1 E).
CEP290 is located in the transition zone
Immunofluorescence microscopy using the antibody directed
against CEP290 showed CEP290 immunoreactivity at the base
of each flagellum of wild-type cells (Fig. 2 A, a–c; and Fig. 2 B);
the label was missing from cep290 mutant cells (Fig. 2 A, d–f),
indicating that the labeling was specific. This localization pat-
tern was confirmed by using the CEP290-HA rescued strain
and antibodies to HA (see Fig. 6). Co-labeling with antibodies
to polyglutamylated tubulin, which label basal bodies and fla-
gellar axonemes but fail to label the transition zone of flagella
(Lechtreck and Geimer, 2000), revealed that the CEP290 signal
was located in the region that lacked polyglutamylated tubulin
labeling, indicating that CEP290 is located in the transition zone
(Fig. 2 C). CEP290 remained associated with the transition zone
after deflagellation (Fig. S1), a finding that is consistent with its
presence in the C. reinhardtii centriole proteome (which included
the transition zone; Keller et al., 2005) and its absence in the
C. reinhardtii flagellar proteome (which lacked the transition
zone; Pazour et al., 2005). CEP290 was undetectable in West-
ern blots of isolated flagella (Fig. S2), which confirmed that
CEP290 does not enter the flagellar compartment distal to the
cep290 mutant cells have defects in the
structures that bridge the transition zone
microtubules and membrane
To determine if loss of CEP290 resulted in ultrastructural defects,
we examined cep290 mutant cells by EM. We observed no defects
in basal bodies. In longitudinal sections through the transition
zone, the wedge-shaped structures that extend between the dou-
blet microtubules and membrane in wild-type cells (Ringo, 1967)
were missing or collapsed onto the doublets in the mutant cells
(Fig. 3, A and B). In cross sections through transition zones, the
Y-shaped connectors that extend from the transition zone doublet
microtubules to the flagellar membrane were often absent in the
mutant cells (Fig. 3, C and D). Image averaging (Fig. 3, C and D,
insets) revealed remnants of Y connectors on the mutant doublets,
including a small electron density possibly corresponding to the
base of the Y connector. In short flagella, we occasionally observed
bulges in the flagellar membrane filled with electron-dense material
(Fig. 3, E–G). In rare instances, we observed defects in axonemal
microtubules (Fig. 3 F); however, the vast majority of sections
olfactory sensory neurons of the rd16 mouse, which harbors
a hypomorphic in-frame deletion in Cep290 and displays early-
onset retinal degeneration and anosmia (Chang et al., 2006;
McEwen et al., 2007). In addition, RNAi knockdown of CEP290
in cultured mammalian cells decreased the percentage of cells
displaying primary cilia (Graser et al., 2007; Tsang et al., 2008).
CEP290 binds to and activates the transcription factor ATF4,
a protein implicated in renal cyst formation (Sayer et al., 2006).
Collectively, the data suggest that CEP290 is involved in ciliary
assembly and the expression, targeting, or transport of ciliary
proteins, but why CEP290 deficiency causes defects in cilia
To elucidate the molecular role of CEP290, we used
Chlamydomonas reinhardtii, which has numerous character-
istics that make it an ideal model organism for studying the
function of ciliary and basal body components. Human and
C. reinhardtii CEP290 are highly conserved (Basic Local
Alignment Search Tool [BLAST] E = 5 e27); Keller et al.
(2005) previously identified the C. reinhardtii CEP290 ortho-
logue by proteomic analysis of isolated centrioles. Our results
show that C. reinhardtii CEP290 is localized to the transition
zone between the flagellar basal body and axoneme, where it is
required for the assembly of the microtubule–membrane link-
ers characteristic of this poorly understood region. Loss of the
protein results in defects in flagellar composition, including alter-
ation of the normal balance of intraflagellar transport (IFT) com-
plexes A and B and abnormal levels of the membrane-associated
proteins BBS4 and PKD2, the human homologues of which are
involved in ciliopathies. The results also show that CEP290 is
highly dynamic at the transition zone, which suggests that it
may be involved in signaling between the cilium and cell body,
and which has clinical implications for CEP290 gene therapy in
the retina to treat LCA due to defects in CEP290. We propose
that CEP290 is part of a complex that links the membrane to
the microtubules in the transition zone and regulates entry of
proteins into the ciliary compartment.
Identification of a C. reinhardtii
To understand how CEP290 deficiency affects flagellar func-
tion, a library of C. reinhardtii insertional mutants was screened
using PCR with primers complementary to sequences in the
5 and 3 regions of the C. reinhardtii CEP290 homologue (pre-
viously designated POC3; Keller et al., 2005). In one strain
(Y168), primers directed toward the 3 end of CEP290 failed
to amplify a product, which indicated that this region is deleted
(Fig. 1 A). Further analysis by PCR revealed an 18.5-kb dele-
tion encompassing all but the first 2–4 exons (out of a total of
37 exons) of CEP290. The mutant cells were mostly palmel-
loid (i.e., failed to hatch from the mother cell wall after mitosis;
Fig. 1 B). Release from the mother cell wall by treatment with
autolysin enzyme (Harris, 2009) demonstrated that the cells have
very short/stumpy flagella, some of which begin to elongate over
time (Fig. 1 C). Occasionally, bulges were present in the short or
stumpy mutant flagella (Fig. 1 D).
939CEP290 function in the flagellar transition zone • Craige et al.
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We thank Drs. R. Bloodgood (University of Virginia, Charlottesville, VA), D. Cole
(University of Idaho, Moscow, ID), B. Eddé (Centre National de la Recherche
Scientifique, Montpellier, France) K. Huang (Yale University, New Haven, CT),
and H. Qin (Texas A&M University, College Station, TX) for kindly providing
antibodies used in this study. We are grateful to D. M. Sanderson (University of
Massachusetts Medical School) for the loan of microscopy equipment.
This research was supported by National Institutes of Health grants
(GM030626 to G.B. Witman, GM087848 to B. Craige, and GM014642
to J.L. Rosenbaum), the Grousbeck Family Foundation (to G.B. Witman and
J.L. Rosenbaum), and the Robert W. Booth Endowment (to G.B. Witman).
G.B. Witman is a member of the University of Massachusetts Medical School
Diabetes and Endocrinology Research Center (DERC; DK32520), and core
resources used in this research were supported by a DERC grant (DK32520).
Submitted: 16 June 2010
Accepted: 11 August 2010
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