517
Sorting and transport in C. elegans: a model system with a
sequenced genome
Sandhya P Koushika* and Michael L Nonet†
In the past few years, yeast and cultured cells have been the
model systems of choice for the study of protein sorting and
transport. Recently, there has been a surge in research in
these areas in Caenorhabditis elegans, with advances in
experimental techniques and genomics. New in vivo assays
that monitor endocytosis and neuronal transport have been
used to delineate roles for several genes in these processes.
Addresses
Department of Anatomy and Neurobiology, Box 8108, 660 South
Euclid Avenue, Washington University School of Medicine, St. Louis,
Missouri 63110, USA
*e-mail: [email protected]
† e-mail: [email protected]
Current Opinion in Cell Biology 2000, 12:517–523
0955-0674/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
AP
adaptor protein
Ce
Caenorhabditis elegans
chc
clathrin heavy chain
che
abnormal chemotaxis
dhc
dynein heavy chain
DIC
differential interference contrast
dnc
dynactin complex components
EST
expressed sequence tag
GFP
green fluorescent protein
IFT
intraflagellar transport
klp
kinesin-like proteins
LDLs
low density lipoproteins
odr
odourant response abnormal
osm
osmotic avoidance abnormal
RME
receptor-mediated endocytosis
RNAi
RNA interference
sel
suppressor/enhancer of lin-12
SV
synaptic vesicle
unc
uncoordinated
vab
variable abnormal morphology
zen
zygotic epidermal enclosure defective
Introduction
Targeting to the correct subcellular compartment is often
required for appropriate protein function. The processes
that regulate these events are referred to as sorting. The
cargo being sorted from the ER, Golgi or other compartments often end up in transport vesicles that are used to
direct them to their destination. The journey of these vesicles and organelles is enabled by intracellular transport.
Although a few mutations in genes involved in sorting
have been identified in Caenorhabditis elegans and
Drosophila, systematic screens to identify sorting components have not been carried out in these genetically
amenable multicellular model systems. This review discusses recent studies on protein sorting and intracellular
transport that highlight the strengths of C. elegans as a
model system.
We summarize the recent development of in vivo cell biological tools in C. elegans that have resulted in the ability to
directly address questions in sorting and transport. This
endeavour is helped by knowledge of the genome
sequence, which is a significant advantage in identifying
members of protein families known to have important sorting and transport functions in other organisms [1]. We
discuss two sorting processes: receptor-mediated endocytosis (RME) and sorting at the plasma membrane. The former
process is being systematically studied using a newly developed in vivo RME assay. This assay assigns roles to C. elegans
orthologues known to function in sorting pathways in other
organisms. Genes with roles in protein sorting at the plasma
membrane have been identified in developmental and
behavioural screens. Also discussed are a subset of proteins
involved in intracellular transport, namely the kinesin and
dynein superfamilies. An in vivo real-time sensory neuron
transport assay assigns a role for some of these molecules in
intraflagellar and dendritic transport.
C. elegans as a model system
Multicellular organisms are composed of intercommunicating specialized cell types with different morphology, polarity
and secretory functions. What are the molecular mechanisms that orchestrate the specialized sorting and transport
requirements of these different cell types? At the genomic
level, cell-type differences can probably be accounted for by
a combination of duplication and functional specialization
within gene families, as well as the evolution of novel gene
families. C. elegans is a useful model system because it is
endowed with the complexity of a multi-cellular organism
while retaining relative simplicity in terms of cell number
and morphology. C. elegans has approximately 940 somatic
cells unlike the other simple genetic model system,
Drosophila, that has over three billion cells.
Amongst the cells of C. elegans are some that are particularly suited to cell biology, including oocytes, coelomocytes,
excretory cells, neurons and gland cells. The oocyte is a
valuable cell type to study RME as nutrients must be
loaded into it for successful independent development of
the zygote. In contrast, the six scavenger coelomocytes
may provide an excellent system for non-RME or phagocytosis. In adults, these non-motile cells have large
vacuoles, many coated pits and vesicles [2]. They can also
accumulate several dyes injected into the pseudocoelom,
suggesting that they function analogously to primitive
phagocytic immune cells [2]. Another cell type in which
sorting to the apical domains can be studied is the excretory cell — the largest mononucleate and polarized cell type
in C. elegans [3]. A beginning has been made in the study of
excretory cell function by isolating mutants that are defective in the apical membrane domain [4]. Some of these
518
Membranes and sorting
Figure 1
(i) Wild type
(ii) Ce-rab7(RNAi)
(a)
Yolk protein–GFP
b
a
(b)
Motor–GFP (cilia)
0s
1s
2s
1s
2s
Cargo–GFP (dendrite)
0s
Anterior
(a) Yolk protein–GFP fusion in (i) wild type and
(ii) rab7(RNAi) oocytes. With rab7(RNAi), the
fluorescence is distributed in larger vesicles
compared to wild type. The brackets indicate
the position of a nearly full grown oocyte. Scale
bar: 5 mm. Figure courtesy of B Grant and D
Hirsh (Columbia University College of
Physicians and Surgeons, USA). (b) Real-time
fluorescence assay using CeKAP–GFP (upper
panel), a component of the kinesin-II complex
and OSM-6–GFP (lower panel), a cargo of
kinesin-II. CeKAP–GFP images were taken in
cilia of the sensory neuron whereas OSM6–GFP images were taken in the dendrite. The
line drawing outlines the morphology of a
chemosensory neuron and arrows indicate the
direction of transport. C, cilia; TZ, transition
zone; D, dendrite; CB, cell body; A, axon. Scale
bar: 5 mm. Figure courtesy of D Signor and J
Scholey (University of California Davis, Davis).
Current Opinion in Cell Biology
mutants may disrupt protein sorting events, thereby altering either the establishment or maintenance of the
polarized epithelium. Therefore, these cells, which maintain the osmotic balance in the animal, provide means to
address cell-type specific sorting [5].
Neurons are specialized secretory cells with process extensions that require long distance transport from the cell body
to the synapse. Serious impairment of neuronal function in
C. elegans, for example by severely reducing neurotransmission, does not compromise viability [6]. Indeed, as few as
three neurons may be required for organismal viability [7].
The gland cells are similar to neurons by virtue of their long
cytoplasmic extensions [2]. They are thought to aid molting
by secreting substances present in large vesicles that loosen
the cuticle. The movement of these vesicles is easily visualized, making these cells an excellent system to address
transport questions [8,9•]. With several cell types and true
multicellularity, C. elegans offers a complex yet simple experimental system to understand basic biological processes.
A major advantage of C. elegans is the ability to isolate
mutants that disrupt particular processes. Using identified
mutants, other genes in the pathway can also be isolated by
secondary suppressor or enhancer genetic screens. A significant development in the study of C. elegans is the use of
reverse genetics technique: introduction of double-stranded RNA interferes (RNAi) with native gene function,
mimicking either a strong loss-of-function or null phenotype [10]. Furthermore, gene knock-outs can also be
generated [11,12]. A key benefit for cell-biological studies
is the transparent cuticle that allows visualization of all
cells, fluorescently tagged proteins and vital dyes within
the living organism. Added to these is the effective completion of the genome sequence [1]. Studies in C. elegans
can be used to augment and advance understanding
obtained from other model systems.
Sorting components
Protein sorting takes place largely in transit through the
secretory and endocytic pathways. A vast number of proteins regulate these pathways including vesicle coat protein
complexes [13], GTPases [14], SNAREs [15] and adaptor
proteins (APs). The majority of these proteins are represented in C. elegans by single orthologues (Table 1).
However, for the AP complex, there are four adaptin genes,
compared with six in S. cerevisiae. The adaptor complex is
composed of two adaptins: a medium chain (m) and a small
chain (s) (Table 1). This strongly suggests that the a and b
genes function as the adaptins for both the AP1 and AP2
complexes. Diversity in the medium and small subunits is
greater: there are two m1 genes and unique m2, m3, s1, s2
Sorting and transport in C. elegans Koushika and Nonet
519
Table 1
Select proteins involved in sorting and transport in C. elegans.
Protein names
Sorting components
Clathrin heavy chain
Clathrin light chain
AP complexes
a-adaptins: a1/2†, d
b-adaptins: b1/2‡, b3
medium chains: m1, m2, m3
small chains: s1, s2, s3
COPI complex
COPI: a, b, b¢, g, d, e, z, ARF1
COPII complex
COPII: Sec13p, Sec31p
Sec23p, Sec24p, Sar1p
p24 family proteins
Dynamin family
Rabs
T-SNAREs
Syntaxin, SNAP-25, Bos, Bet families
V-SNAREs
Synaptobrevin
Number of genes*
Mutants
RNAi etc.
1
1
–
–
Yes
–
1, 1
1, 1
2, 1, 1
1 each
–
–
unc-101 (m1), dpy-23(µ2)#
–
a1/2
b1/2
m2
s2
1 each
–
b¢, z, arf1
1 each
–
–
4
3
at least 16
sel-9
dyn-1
rab-3 [23]
sel-9, one other
dyn-1 [48], drp-1 [49]
rab5, rab7, rab11
at least 15
unc-64 [58], ric-4§
–
at least 8
snb-1 [33]
–
22
2
1
1
unc-104, unc-116, osm-3, zen-4, vab-8
–
–
che-3
klp-3‡‡, zen-4
–
Yes
–
Transport components
Kinesin-like heavy chains**
Kinesin-light chains
Cytoplasmic dynein-heavy chain
DHC1b
*Although an attempt has been made to be accurate, some genes and
widely divergent family members may not be listed. †a1/2 indicates the
a subunits of both the AP1 and AP2 complexes. a1 is also known as g
[13]. ‡b1/2 indicates the b subunits of both the AP1 and AP2
complexes. #dpy-23 encodes the µ2 intermediate chain of AP2
(G Garriga, personal communication). §ric-4 encodes SNAP-25 (J Lee,
Y Lee, ML Nonet, J Rand, B Meyer, unpublished data). **Most klps are
listed in the kinesin homepage (www.blocks.fhcrc.org/~kinesin) [29].
‡‡The klp-3 mutant phenotype was ascertained by expressing antisense
klp-3 [41]. Individual members of a protein family are referenced in
either the text or the table.
and s3 genes [16] (Table 1). Since most structural components implicated in the endocytic and secretory pathways
are present in a single copy, isolation and analysis of
mutants lacking these constituents should be feasible.
released into the pseudocoelom and subsequently endocytosed by the oocyte [18,19•]. Amongst other constituents,
yolk is composed of a protein known as vitellogenin, which
is homologous to ApoB-100, a component of low density
lipoprotein (LDL) particles [20]. Yolk RME probably proceeds along pathways that are similar to the LDL-receptor
internalization pathways in mammals [17]. An assay in living animals for yolk RME has been developed using green
fluorescent protein (GFP) fused to vitellogenin. This
fusion protein reports the transport of yolk protein into
C. elegans oocytes (Figure 1A) [18•].
The greater diversity of secretion and transport requirements in multicellular organisms, in comparison to
S. cerevisiae, suggests an increased multiplicity of some
components of these pathways. Commensurately, the
diversity of genes encoding members of the rab, SNARE
and kinesin families is markedly increased (Table 1). This
fits well with the current view of these families in regulating specific aspects of sorting, docking, fusion and vesicle
transport in different cell types. Despite C. elegans being
vastly more complex than yeast, the diversity in these protein families still pales in comparison to vertebrates. From
our perspective, it is the study of the more complex protein
families in C. elegans that offer the greatest promise for dissecting the molecular mechanisms underlying distinct
secretory/transport pathways.
Receptor-mediated endocytosis
Sorting from the plasma membrane by RME is a widely
used mechanism for uptake of components such as
yolk [17]. In C. elegans, yolk is synthesized in the intestine,
The RME assay was used to define roles for orthologues of
genes identified by RNAi [18•]. Strong inhibition of yolk
uptake and inviable embryos result from RNAi of clathrin
heavy chain (chc), dynamin-1 (dyn-1), a-adaptin and
b-adaptin. In contrast, µ2(RNAi), s2(RNAi) or µ2(RNAi)
in a µ1 mutant (unc-101) background did not affect yolk
uptake or viability. However, the authors did not test the
effect of mutating the alternate m1 subunit, which may be
able to compensate for the loss of both m2 and unc-101
(Table 1). RNAi phenotypes of some rabs were striking:
rab5 inhibited yolk uptake, rab11 reduced yolk uptake and
rab7 redistributed yolk into a few larger sized vesicles at
the periphery of oocyte but did not inhibit uptake
520
Membranes and sorting
(Figure 1a). Although the authors did not resolve whether
AP1 or AP2 are involved in yolk endocytosis, they validated their assay by demonstrating a role for proteins involved
in RME in other systems. RNAi of some secretory pathway
components — namely §’-COP, z-COP and arf-1 resulted
in severe phenotypes that led to oocyte disintegration.
The phenotypic differences between perturbing the endocytic and secretory pathways should be helpful in
identifying new RME mutants.
To identify additional components of the RME pathway,
mutations in twelve genes (rme) were isolated in a forward
genetic screen using the in vivo RME assay [18•]. Of these,
rme-2 encodes the yolk receptor belonging to the LDL
super family, which is necessary and sufficient for yolk protein uptake. Distribution of the yolk and RME-2 proteins
in chc(RNAi), rab5(RNAi) and rab11(RNAi) oocytes
showed that they behave like a receptor–ligand pair. The
results with RNAi of certain AP components, along with
the isolation of several new mutants, will presumably
explain how endocytosis in C. elegans oocytes differs from
established pathways identified in yeast and mammalian
cells. Furthermore, cloning of other rme mutants may identify novel constituents and regulators of the pathway.
Sorting to the plasma membrane
No specific assay has been developed to examine sorting at
the cell surface, but analysis of two mutants — sel-9 and
unc-11 — suggests that they influence this process. A suppressor screen with a morphological phenotype of a Notch
family member, lin-12 yielded several suppressors including sel-9 and sel-12 (a presenilin) [21,22]. sel-9 encodes a
p24 family member, one of four in C. elegans, which are
major components of the COPI and COPII vesicles [23•].
All isolated sel-9 alleles show genetic interactions with several different missense mutations affecting the LIN-12
extracellular domain, but none affected the phenotype of a
lin-12 null allele. In addition, the sel-9 alleles also suppress
certain missense mutations in another Notch family member, glp-1. The basis of suppression of the glp-1 phenotype
appears to be the increased cell surface expression of
mutant GLP-1 in sel-9 compared to wild type, where the
mutant protein is retained intracellularly. Without knowing
the intracellular localization of SEL-9, it is difficult to
determine the organelle in which this protein acts.
However, SEL-9’s role appears to be intrinsically linked to
quality-control mechanisms, so that the loss of its function
allows mutant proteins to escape to the cell surface [23•].
This role is in keeping with the proposed functions of the
p24 family proteins, such as in cargo selectivity and recruitment of coat proteins, in other systems [13].
Work on the AP180 orthologue, unc-11, identified a novel
role for this gene in protein sorting [24]. In unc-11 mutants,
synaptobrevin, a synaptic-vesicle (SV) protein, is mislocalized, probably to the plasma membrane. Other SV proteins
are, however, efficiently targeted to their appropriate compartment [24]. This phenotype was surprising as AP180 was
initially thought to be involved primarily in endocytosis, and
AP180 mutants in both C. elegans and Drosophila have a role
in regulating vesicle size [24,25]. unc-11 was the first mutant
identified that affects the sorting of a SV protein; the signals
and partners required for sorting other SV proteins are, as
yet, unknown. C. elegans awaits systematic identification of
genes that regulate sorting at the plasma membrane.
Known roles of kinesin-like proteins
Once proteins and lipids are sorted into transport vesicles
from the donor compartment they frequently have to traverse the cell to reach their target destination. Such long
distance transport often requires the microtubule-dependent molecular motors of the kinesin and dynein
superfamilies [26]. Transport along the axon is one key
microtubule motor-dependent process. One can visualize
fluorescently tagged motors/cargo in C. elegans neurons and
thereby assess neuronal transport. This is particularly useful in mechanosensory neurons, which have unique large
diameter microtubules [27]. Their absence leads to a block
in transport but results in no more severe phenotype than
the the lack of response to gentle-touch [28].
There are at least 22 kinesin-like (klp) heavy chain genes in
C. elegans with representatives from all major subfamilies [26,29]. This is in contrast to Saccharomyces which has
only six klps whereas mice express at least 38 [29]. Although
forward genetics has identified mutants in some klps, the
roles of most of these genes remain to be investigated.
The mutant phenotypes and potential cargoes of some
well defined klps are described below. The cargo of
UNC-104 was revealed by electron microscopy analysis,
which showed very few SVs at synapses, instead vesicles
accumulated in the cell body [29]. This has been subsequently borne out by several studies showing that
SV-associated proteins mislocalized to the cell body in
unc-104 mutants [31–34]. Phenotypes of mutants in the
conventional kinesin-heavy chain gene unc-116 include
maternal-effect lethality, which is caused by defects in the
first cleavage events, mispositioning of axons, absence of
rough ER and shorter body length [35,36]. In neurons,
unc-116 mutants mislocalize mitochondria, as visualized by
organelle-targeted GFP (SP Koushika, unpublished data).
In vivo, UNC-116 presumably functions in association with
the kinesin-light chains, of which there are two representatives in the genome [29]. These pleiotropic phenotypes
demonstrate that UNC-116 probably plays widespread
roles. The mammalian orthologues of UNC-104 and
UNC-116, KIF1A and KIF1B, have roles in SV transport,
lysosome and mitochondrial dispersion respectively
[37,38]. Another klp is osm-3, and animals lacking this gene
have defects in various sensory perceptions [39,40]. Two
other klps, klp-3 and zen-4, function in chromosome segregation and cytokinesis; these roles are similar to their
orthologues Ncd/Kar-3 and MKLP-1, respectively, in other
model systems [41–43]. Although the demonstrated function of the two above klps are similar to their orthologues,
Sorting and transport in C. elegans Koushika and Nonet
the role of klp-3 and zen-4 in other intracellular transport
events has not yet been investigated.
A divergent member of the klps is encoded by one of the
transcripts of vab-8 (VAB-8L), which has homology with
the kinesin-motor domain [44]. It remains to be determined whether the motor domain is capable of binding
microtubules and hydrolyzing ATP. Nevertheless, VAB-8L
has a role in global control of posteriorly directed growth
cone migrations and certain posteriorly directed cell migrations [44]. This gene provides a good example of novel
functions that may be uncovered for other klps where
C. elegans offers a simple multi-cellular organism to address
the role of multiple kinesin-like genes in transport.
Known roles of the cytoplasmic dynein
super-family
The C. elegans genome has at least two cytoplasmic dyneinheavy chain genes. The dynein-heavy chains function as a
homodimer along with the dynactin complex [26]. So far,
no conventional mutants have been reported for the cytoplasmic dynein-heavy chain (dhc-1) orthologue in
C. elegans [45]. However an early role for dhc-1 in cell division in the one-cell embryo has been demonstrated by
RNAi [46]. Time-lapse differential interference contrast
(DIC) imaging of cell division has shown that a large
reduction in DHC-1 results in a lack of migration of the
male and female pronuclei, failure to separate the centrosomes and separation of the centrosomes from the male
pronucleus. However, due to the lethal effects of
dhc-1(RNAi) its role in the later stages of development, for
instance in axonal transport or maintenance of the Golgi,
remain to be investigated. The Golgi is highly vesiculated
and lysosomes are mispositioned in cells lacking mammalian cytoplasmic DHC [47]. RNAi of the dynactin
complex components dnc-1 (p150 Glued) and dnc-2 (dynamitin) also gives rise to similar early embryonic cell
division phenotypes [48]. The cytoplasmic heavy chain
gene che-3 belongs to the DHC1b subfamily, which phylogenetically lies between the cytoplasmic and axonemal
dynein-heavy chain families [49]. Mutations in che-3 result
in defective chemosensation; the protein is expressed only
in ciliated sensory neurons and plays restricted roles in
intracellular transport (see below).
Intraflagellar and dendritic transport in ciliated
sensory neurons
Chemosensation in C. elegans is an important sensory function mediated by several ciliated neurons that are open to
the environment [40]. To recognize chemical stimuli it is
essential to assemble and maintain axonemal components
and signal-transduction machinery at the ciliated endings
of chemosensory neurons. The kinesin-II complex drives
anterograde intraflagellar transport (IFT) in Tetrahymena
and Chlamydomonas cilia. It also functions as an axonal
motor [50,51]. The heterotrimeric kinesin-II complex in
C. elegans consists of CeKRP85, CeKRP95 and CeKAP, all of
which have been identified either from the genome
521
sequencing or EST projects [52]. All three proteins are
expressed in chemosensory neurons and are highly concentrated at the tips of the sensory cilia, probably at their
ciliated endings [52]. In Chlamydomonas, IFT polypeptides
are the cargo of kinesin-II [50]. osm-1 and osm-6 are C. elegans genes that encode proteins homologous to the IFT
polypeptides in Chlamydomonas [53,54].
A significant step forward in the study of transport in C. elegans has been the development of a real-time fluorescence
assay using GFP fusion proteins [55•,56•] (Figure 1B). The
anterograde and retrograde velocities of CeKAP, OSM-6
and OSM-1 along ciliated endings and the dendrite are
remarkably similar, suggesting that they are transported by
the same motor, most probably kinesin-II [56•]. In contrast, a sensory ciliary transmembrane receptor, ODR-10,
moves at a faster anterograde rate, suggesting that it is
transported by a different motor [55•]. Although mutations
in the kinesin-II complex would demonstrate a
motor–cargo relationship, no such mutants have been
described. OSM-3, another motor protein expressed in ciliated neurons, does not alter the transport of any of the
GFP-tagged proteins described above [39,52,56•].
Mutations in the DHC1b orthologue, che-3, on the other
hand, affects the retrograde IFT of OSM-6 and CeKAP
[56•]. Anterograde transport, and dendritic transport of the
cargo components tested, were unaltered in che-3 mutants.
This highlights not only the restricted function of che-3 in
a subcompartment of the dendrite, but also that other
unidentified retrograde motors play a role in dendrites.
CHE-3 may also play a role in recycling of CeKAP and
other components for use in another transport cycle.
Although a few motors have been identified, the modulators of dendritic transport are still unknown. ODR-4, a
novel membrane-associated molecule that mediates the
localization of odourant receptors is a candidate regulator
of this process [57]. The IFT transport assay, along with a
large number of viable mutants defective in chemosensation, offers an attractive system to identify many
modulators of dendritic and ciliary transport.
Conclusions
The general mechanism of intracellular sorting and transport is reasonably well established in model organisms. In
C. elegans, assays have been developed to study distinct
intracellular sorting and transport processes. Using these
assays, the cellular and organismal roles of sorting components are being delineated. A major step forward will be
the identification of new constituents and regulators of the
basic pathways, which will draw upon both the genetic
amenability of the organism and in vivo assays. Comparison
of sorting in various cell types will elucidate how the general pathway is modified to accommodate the
requirements of different cell types. Many facets of regulation of distinct sorting and transport pathways remain
obscure. For instance, how is the level of protein sorted to
different compartments controlled? How are motor proteins transported to their site of action? How is the release
522
Membranes and sorting
of cargo at specific sites achieved? In vivo analysis in genetically tractable systems should provide one means of
integrating the findings of genetic, molecular and biochemical studies in our quest to understand these complex
cellular processes. Progress in C. elegans presages the likely
contributions from in vivo studies of sorting and transport
in a variety of model organisms as sequencing of entire
genomes facilitates their study.
Note added in proof
The genome of Drosophila was published after this review
was written and a catalogue of members of some families
involved in aspects of sorting and transport has been
made [59,60]. This model system offers some of the same
advantages as C. elegans in addressing questions related to
protein sorting and intracellular transport.
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We thank Kathryn Miller, Dorothy Schafer, Chris Kaiser and members of
the Nonet and Salkoff laboratories for valuable suggestions. We thank Barth
Grant, David Hirsh, Dawn Signor and Jonathan Scholey for providing
figures at short notice. Research in MLN’s laboratory is supported by grants
from USPHS. SPK is supported by a fellowship from McDonnell CCMN.
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·
Caenorhabditis elegans oocyte. Mol Biol Cell 1999, 10:4311-4326.
Grant and Hirsh develop an in vivo assay to monitor yolk-protein endocytosis in oocytes. Using RNAi, the authors demonstrate a role for orthologues
of several proteins involved in the secretory and endocytic pathway in other
organisms. Among several isolated mutations in genes regulating endocytosis, they cloned one that encodes the yolk protein receptor. The yolk receptor and its ligand behave as a pair when protein traffic is disrupted by RNAi.
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