MINI REVIEW ARTICLE
published: 07 August 2013
The mysterious case of the C. elegans gut granule: death
fluorescence, anthranilic acid and the kynurenine pathway
Cassandra Coburn and David Gems*
Institute of Healthy Ageing, and Department of Genetics, Evolution and Environment, University College London, London, UK
Elena G. Pasyukova, Institute of
Molecular Genetics of the Russian
Academy of Sciences, Russia
Di Chen, Nanjing University, China
Shin Murakami,Touro University
Arjumand Ghazi, University of
Pittsburgh School of Medicine, USA
David Gems, Institute of Healthy
Ageing, and Department of Genetics,
Evolution and Environment, University
College London, Gower Street,
London WC1E 6BT, UK
Gut granules are lysosome-like organelles with acidic interiors that are found in large
numbers within the intestine of the nematode Caenorhabditis elegans.They are particularly
prominent when viewed under ultraviolet light, which causes them to emit intense blue
fluorescence.Yet the function of these large and abundant organelles in this heavily-studied
model organism remains unclear. One possibility is that they serve as storage organelles,
for example of zinc. A new clue to gut granule function is the identification of the blue
fluorescent material that they contain as a glycosylated form of anthranilic acid, which is
derived from tryptophan by action of the kynurenine pathway. This compound can also
serve a surprising role as a natural, endogenous marker of organismal death.
THE GUT GRANULE: AN ENIGMATIC NEMATODE ORGANELLE
Despite decades of research on the nematode Caenorhabditis
tion of the prominent organelles known as gut granules, which
are numerous in the intestinal cells of nematodes throughout the
suborder Rhabditina (Chitwood and Chitwood, 1950). A strik-
ing feature of gut granules is the blue fluorescence that they emit
under ultraviolet light (Klass, 1977; Gerstbrein etal., 2005). Clues
to gut granule function include their acidic interior and capac-
ity for endocytosis (Clokey and Jacobson, 1986; Hermann etal.,
2005),both lysosome-like features (though gut granules are much
bigger than normal lysosomes). This and the fluorescent material
within identify gut granules as lysosome-like organelles (LROs;
Hermann etal., 2005; Bernabucci etal., 2012), akin to pigment-
containing melanosomes in mammals and eye pigment granules
in Drosophila (Raposo and Marks,2007). Thus,the identity of the
blue fluorescent substance could provide a key to understanding
gut granule function.
One suggestion is that the source of gut granule fluorescence is
lipofuscin, a complex molecular waste production that accumu-
Lipofuscin can contain Schiff bases, which have similar spectral
similarities to the worm blue fluorescence (Fletcher etal., 1973;
in aging worm populations (Klass, 1977; Davis etal., 1982; Gerst-
brein etal.,2005).Another idea,derived from studies of C.elegans
Flu mutants with altered fluorescence color and intensity, is
that the blue fluorescence emanates from L-tryptophan-derived
metabolites called kynurenines (Babu, 1974).
Over the years the lipofuscin interpretation has been favored
(see e.g., Gill, 2006; Masse etal., 2008; Fujii etal., 2009; Jain etal.,
Unfortunately, this interpretation (i.e., that the blue fluorescent
substance is lipofuscin) is not the correct one.According to recent
chemical analysis, the fluorescent substance within gut granules
is a kynurenine pathway product, anthranilic acid (AA) glucosyl
ester(Coburn etal.,2013),consistent with the proposal of P. Babu
and S. S. Siddiqui so many years ago (Babu, 1974; Bhat and Babu,
1980; Siddiqui and Babu, 1980). This chemical identification was
lack gut granules (Hermann etal., 2005). Whether or not lipofus-
cin exists in C. elegans remains an open question. Thus,C. elegans
gut granules contain large quantities of AA. But what it is there
for? Here, one may seek clues from kynurenine pathway action in
THE KYNURENINE PATHWAY AND NEURODEGENERATION
In mammals, the kynurenine pathway generates a variety of
important molecules, including the co-factor nicotine adenine
dinucleotide (NAD) and the neurotransmitter serotonin. Around
95% of tryptophan (the rarest essential amino acid) is consumed
by this pathway (Vecsei etal., 2013). Although discovered over
150 years ago, the action of the kynurenine pathway’s inter-
mediate metabolites, known as kynurenines, has until recently
been relatively little studied (Schwarcz etal., 2012). One role
of kynurenines is in modulating CNS excitability (Perkins and
Stone, 1982; Hilmas etal., 2001; Vecsei etal., 2013). For example,
the kynurenine quinolinic acid stimulates N-methyl-D-aspartate
(NMDA) receptors (Stone and Perkins, 1981; Schwarcz etal.,
2012), while kynurenic acid antagonizes all excitatory amino acid
Kynurenine pathway dysregulation has been implicated in
neurological disorders, including Huntington’s, Alzheimer’s, and
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Coburn and GemsGut granules, death fluorescence and kynurenines
Parkinson’s disease, multiple sclerosis, and epilepsy (Vecsei etal.,
2013) as well as in neurodegeneration caused by acute insults,
such as ischemia and excitotoxicity (Stone etal., 2012). Excito-
toxic neurodegeneration is caused by release of high levels of
excitatory neurotransmitters, which trigger an influx of calcium
ions after depolarization (Rothman and Olney, 1987). Thus, cal-
cium can act as a second messenger, triggering the initiation of
necrotic cell death (Rothman and Olney, 1995). The kynurenine
quinolinic acid can act as an excitotoxin: levels increase following
ischemia, and correlate with increased neurodegeneration (Saito
etal., 1993). Thus, one of the ways in which kynurenines may
contribute to neurodegenerative disease is by inducing excitotoxic
THE KYNURENINE PATHWAY IN C. elegans
Is there a link between kynurenines and aging, particularly neu-
rodegeneration, in C. elegans? Very little is known about the
biology of kynurenines in nematodes. One exception relates back
to gut granules: among the Flu mutants alluded to previously,
altered intestinal fluorescence (Flu) phenotypes can arise from
mutations affecting kynurenine pathway enzymes. For example,
flu-1 mutants, which show an altered, bluish-purple gut granule
fluorescence, have reduced kynurenine-3-hydroxylase activity
(Siddiqui and Babu, 1980), and flu-2 mutants, which show a dull
green fluorescence, have reduced kynureninase (Bhat and Babu,
1980; Figure 1A). The C. elegans genome contains homologs of
genes encoding these two enzymes in the vicinity of the flu-1 and
flu-2 loci: a kynurenine hydroxylase, R07B7.5, and a kynureni-
nase C15H9.7, respectively (Altschul etal., 1990; Kanehisa, 2012).
Other predicted kynurenine pathway genes are also present in C.
elegans (van der Goot and Nollen, 2013).
In Drosophila genetic and pharmacological inhibition of the
kynurenine pathway enzyme tryptophan 2,3-dioxygenase (TDO)
extends longevity (Oxenkrug, 2010; Oxenkrug etal., 2011). This
suggests that kynurenines may contribute to pathologies of aging;
however, whether this is true in C. elegans remains uncer-
tain. Here RNAi knock-down of tdo-2 reduced the toxicity of
α-synuclein aggregation in a Parkinson’s disease model, and
increased lifespan (van der Goot etal., 2012). However, these
effects proved to be caused by increased levels of tryptophan
rather than altered levels of kynurenines (van der Goot etal.,
2012; for a detailed review of the kynurenine pathway and aging
FIGURE 1 | (A) Synthesis of anthranilic acid by the kynurenine pathway.
(B) Death fluorescence in young adult C. elegans killed with a heated wire
(DAPI filter). During death fluorescence the pattern of fluorescence changes
from punctate (issuing from gut granules) to diffuse, and much brighter. A, P ,
anterior and posterior ends of intestine.Time is relative to peak fluorescence.
Scale bar, 200 μm.
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Coburn and GemsGut granules, death fluorescence and kynurenines
seevan der Goot and Nollen, 2013).tdo-2RNAialsoabrogatesgut
granule fluorescence in the worm (Coburn etal., 2013).
Kynurenines also play a startling role in the biology of death
in C. elegans. As they die, worms emit a dramatic burst of blue
AA fluorescence (Coburn etal., 2013; Figure 1B). This death
fluorescence typically occurs in an anterior to posterior wave that
courses along the intestine,and is seen in both young worms sub-
jected to lethal injury, and worms dying peacefully of old age.
Death fluorescence is a somewhat eerie phenomenon in that it
renders visible the passage of death through the semi-transparent
body of the worm as a spectral blue glow.
Death fluorescence is promoted by the calpain–cathepsin
necrotic cell death cascade. In this cascade,intracellular Ca2+lev-
els rise, activating Ca2+-dependent calpains (cysteine proteases;
Yamashima etal., 1996). These cause lysosomal lysis, leading
to cytosolic acidosis and the destructive release of lysosomal
cathepsin proteases (Yamashima and Oikawa, 2009). Mutational
attenuation of this cascade often reduces death fluorescence
(Coburn etal., 2013). Moreover, the intercellular propagation of
death fluorescence (and, probably, necrosis) is dependent upon
the innexin gap junction INX-16, reminiscent of the spread of
excitotoxic neuronal death from one cell to another in mammals.
How exactly the necrotic cascade leads to increased AA fluores-
cence remains unclear, but one possibility is that it reflects AA
fluorescence dequenching as it is released from the gut granules
upon organellar lysis.
POSSIBLE FUNCTIONS OF ANTHRANILATES AND GUT
GRANULES IN C. elegans
The significance of AA concentrated within gut granules remains
unclear. One possibility is that glycosylation of AA contributes
to its accumulation; in Arabidopsis, glycosylation by UDP-
glucosyltransferases promotes AA accumulation by increasing
compound stability (Quiel and Bender, 2003). Regarding func-
kynurenines can contribute to immune function (Munn etal.,
1998; Fallarino etal., 2002; Piscianz etal., 2011). Moreover, AA
can inhibit growth of bacterial pathogens, e.g., Legionella pneu-
mophila (Sasaki etal., 2012). Thus, AA might have antibiotic
store of anti-bacterial agents in the event of pathogen attack. This
suggests a broader role for gut granules: that of chemical weapons
depots for C. elegans in their war against the diverse pathogens
that beset them in their natural environment (Felix and Braendle,
2010). This could also explain the presence of gut granules in the
intestine, the site most likely to experience pathogenic invasion
in C. elegans (Hodgkin and Partridge, 2008). Another possibility,
suggested by similarities between gut granules and melanosomes,
is that they are photoprotective. AA fluorescence (peak λex/λem
340 nm/430 nm) entails the conversion of damaging UV light to
relatively harmless visible light, and so may protect against UV
The large size of gut granules relative to ordinary lysosomes
is consistent with function as a storage organelle. Moreover, gut
granules are the major site of storage of zinc in the worm (Roh
etal., 2012). Interestingly, when zinc levels are high, gut granule
morphology changes, becoming bilobed, including an apparently
non-acidic compartment in which zinc is concentrated. How
distribution of zinc and AA compares in such bilobed gut gran-
ules remains to be established. It is also notable that both metal
toxicity and kynurenines are determinants of neurodegenerative
red; however, results of careful analysis imply that this does not
reflect the presence of lipid within gut granules (O’Rourke etal.,
Ultimately, the role in C. elegans biology of gut granules and
tigation. But we now know at least that the fluorescence of these
prominent organelles issues from AA glucosyl esters, rather than
lipofuscin – removing one reason for believing that worm aging
is caused by accumulation of molecular damage, and opening the
we know that gut granule decay contributes to a wave of intestinal
We thank Alex Benedetto and our referees for comments
on the manuscript. This work was supported by funding from
the Biotechnology and Biological Sciences Research Council,
the Wellcome Trust (Strategic Award) and the European Union
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.
Received: 30 May 2013; accepted: 21 July
2013; published online: 07 August 2013.
Citation: Coburn C and Gems D (2013)
The mysterious case of the C. ele-
gans gut granule: death fluorescence,
anthranilic acid and the kynurenine
pathway. Front. Genet. 4:151. doi:
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