gravity force transduced by the mec-4/mec-10 …...2007/08/24 · nahui kima,b, catherine m....
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Kim et al. Page 1 8/6/07
Gravity force transduced by the MEC-4/MEC-10 DEG/ENaC channel modulates DAF-
16/FoxO activity in C. elegans
Nahui Kima,b, Catherine M. Dempsey c,e, Chih-Jen Kuanc, Jim V. Zovalb, Eyleen O’Rourke d,
Gary Ruvkun d, Marc J. Madou b§ and Ji Y. Sze c§
a Interdisciplinary Materials Science and Engineering
b Department of Mechanical and Aerospace Engineering
University of California, Irvine
Irvine, California 92697
c Department of Molecular Pharmacology
Albert Einstein College of Medicine, Yeshiva University,
Bronx, NY
d Department of Molecular Biology
Massachusetts General Hospital, Boston, MA
e Current address: Dept of Biology, National University of Ireland, Maynooth, Ireland
§ Corresponding authors:
M.J.M (Tel: 949-824-6585, E-mail: [email protected]
J.Y.S (Tel: 718-430-2084, E-mail: [email protected])
Genetics: Published Articles Ahead of Print, published on August 24, 2007 as 10.1534/genetics.107.076901
Kim et al. Page 2 8/6/07
Running title:Regulation of FoxO by DEG/ENaC channel
Key words: gravity, mechanical stress, DEG/ENaC channel, FoxO transcription factor,
metabolism
Corresponding author: Ji Ying Sze, Dept of Molecular Pharmacology, Albert Einstein College
of Medicine, Yeshiva University, 202 Golding Building, 1300 Morris Park Avenue, Bronx, NY
10461. Tel: (718) 430-2084 FAX: (718) 430-8792 E-mail: [email protected]
Kim et al. Page 3 8/6/07
ABSTRACT
The gravity response is an array of behavioral and physiological plasticity elicited
by changes in ambient mechanical force and is an evolutionarily ancient adaptive
mechanism. We show in C. elegans that the force of hypergravity is translated into
biological signaling via a genetic pathway involving three factors: the DEG/ENaC
mechanosensory channel of touch receptor neurons, the neurotransmitter serotonin, and
the FoxO transcription factor DAF-16 known to regulate development, energy metabolism,
stress responses, and ageing. After worms were exposed to hypergravity for 3 hr, their
muscular and neuronal functions were preserved, but they exhibited DAF-16::GFP nuclear
accumulation in cells throughout the body and accumulated excess fat. Mutations in MEC-
4/MEC-10 DEG/ENaC or its partners MEC-6, MEC-7 and MEC-9 blocked DAF-16::GFP
nuclear accumulation induced by hypergravity but did not affect DAF-16 response to other
stresses. We show that exogenous serotonin and the antidepressant fluoxetine can
attenuate DAF-16::GFP nuclear accumulation in wild-type animals exposed to
hypergravity. These results reveal a novel physiological role of the mechanosensory
channel, showing that the perception of mechanical stress controls FoxO signaling
pathways and that inactivation of DEG/ENaC may decouple mechanical loading and
physiological responses.
Kim et al. Page 4 8/6/07
Introduction
The physical force of gravity is a fundamental environmental parameter shaping the life
of biological systems on earth, ranging from unicellular organisms to plants, animals and men.
Musculoskeletal systems, sensory networking and metabolic machinery have evolved to
counterbalance the earth gravitational force of 1G, enabling organisms to maintain posture, grow
and reproduce in the terrestrial environment. Acute or chronic exposure to microgravity
(spaceflight) or hypergravity (centrifugation) generates mechanical stress, which has been shown
to cause organisms to remodel their cellular and physiological processes, including the duration
of exponential growth in the case of bacterium Escherichia Coli (E. coli), the growth rate of
plants, the differentiation features of mammalian tissue cells, and the characteristics of the
musculoskeletal system, endocrine system, body temperature, adiposity and ageing of animals
(FULLER et al. 2002; LE BOURG 1999; MACHO et al. 2001; MOREY-HOLTON 2003; SOGA et al.
2005). The phenotypes induced by changes in the gravitational force environment mostly
disappear after the return to 1G, indicating that living organisms are hard wired to respond to the
ambient physical force and that gravity response is a form of behavioral and physiological
plasticity. Studies from a wide range of fields have identified many critical components
involved in mechanotransduction, such as ion channels, cytoskeletal proteins and signaling
components (INGBER 2006; ORR et al. 2006). However, very little is known about how theses
components contribute to the pathway that translates mechanical stress into cellular mechanisms
to generate physiological modifications in the context of a whole animal.
The FoxO (Forkhead-containing,O subfamily) transcription factors are the best
characterized regulators of stress responses in diverse organisms across phyla (KOPS et al. 2002;
LEE et al. 2003; MCELWEE et al. 2003; ACCILI and ARDEN 2004; MURPHY et al. 2003;
HWANGBO et al. 2004). The transcriptional activity of FoxO is controlled by subcellular
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localization. Signaling from insulin/insulin like-growth factor (IGF)-1 receptors results in
phosphorylation of specific serine/threonine residues of FoxO, thereby sequestering FoxO in the
cytoplasm. Conversely, aversive environmental and physiological cues or mutations abrogating
insulin/IGF-1 receptor signaling cause FoxO nuclear accumulation, where they activate genes
involved in development, energy metabolism, detoxification and immunity, producing enhanced
stress resistance (ACCILI and ARDEN 2004; ANTEBI 2007). In C. elegans, inactivation of the
insulin/IGF-1 receptor DAF-2 either by mutations or aversive factors causes FoxO factor DAF-
16 nuclear accumulation, resulting in developmental arrest, a shift of the metabolic profile
favoring fat deposition, increased expression of a battery of homeostatic stress-response genes
such as heat shock proteins and antioxidant enzymes, and extension of lifespan ( OGG et al.
1997; LIN et al. 1997; KIMURA et al. 1997; HENDERSON and JOHNSON 2001; LEE et al. 2001; LIN
et al. 2001).
The signaling pathway from the DAF-2 insulin/IGF-1 receptor to DAF-16/FoxO is
regulated by neuronal activity (AILION et al. 1999; APFELD and KENYON 1999; WOLKOW et al.
2000; ALCEDO and KENYON 2004). For example, worms bearing defective chemosensory
neurons cannot sense the external chemical world, and these animals exhibit DAF-16 nuclear
accumulation even under optimal growth conditions (LIN et al. 2001). Conversely, applying the
neurotransmitter serotonin or the antidepressant fluoxetine known to increase synaptic levels of
endogenous serotonin attenuates DAF-16 nuclear accumulation in wild-type (WT) animals under
aversive conditions (LIANG et al. 2006). These observations suggest that behavioral and
physiological response to the environment reflects the interplay between neuronal signaling and
the FoxO pathway, and that changes in neuronal activity may modify the stress resistance of the
animal.
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In this study, we describe DAF-16/FoxO response to the mechanical stress of
hypergravity and identify molecular mechanisms underlying this mechanosensory-to-FoxO
transduction. Using a compact disc (CD) based microfluidic cultivation system (KIM et al.
2007), we monitored the effects of hypergravity on neuronal function, muscular structure, and
metabolic profiles in living worms and discovered that MEC-4/MEC-10 degenerin/epithelial
Na+ channel (DEG/ENaC) signaling is required for hypergravity force to induce DAF-16 nuclear
accumulation. The mec-4 and mec-10 genes encode DEG/ENaC proteins and form a
heteromeric, voltage-independent, amiloride-sensitive mechanosensory channel localized along
the axons of six touch receptor neurons that sense gentle mechanical stimuli and mec-10 is
additionally expressed in the PVD neurons that sense strong (harsh) mechanical stimuli
(O’HAGAN AND CHALFIE, 2006). The sensory processes of these neurons extend along the length
of the body. In vivo whole-cell recording has indicated that the MEC-4/MEC-10 channel can be
activated by applying physical force against the body wall (O'HAGAN et al. 2005). Conversely,
mutations in either mec-4 or mec-10 render worms touch insensitive (DRISCOLL and CHALFIE
1991; HUANG and CHALFIE 1994). Our study indicates that mechanosensation does not simply
produce muscular reflex. Our results suggest that signaling from MEC-4/MEC-10 can influence
a broad spectrum of physiological mechanisms, and inactivation of DEG/ENaC signaling of
touch receptors can decouple mechanical loading on the body and physiological mechanisms.
MATERIALS AND METHODS
Strains
C. elegans strains used in this study were: wild-type variety Bristol strain (N2), TJ356(daf-
16::gfp), DR1808(daf-7::gfp), GR1333 (tph-1::gfp), RW1596 stEx30(Pmyo-3::gfp), mec-
6(e1342), mec-9(e1494), mec-10(e1515), mec-7(u88), mec-4(u253), mec-4(e1611), and SK4005:
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zdIs5[mec-4::gfp; lin-15(+)]. Worms were maintained on Nematode Growth Medium (NGM) at
20°C with E. coli OP50 as a food source (BRENNER 1974).
100G paradigm
The design and fabrication of the C. elegans CD cultivation system, including the materials, the
microfluidic system, the dimensions of the chambers, and the spin-stand system used to apply
centrifugal force have been detailed described previously (KIM et al. 2007). The main
fabrication material of the microfluidic platform is Polydimethylsiloxane (PDMS), which has the
advantage of biocompatibility, high permeability to gases, chemically inert, and optical
transparency down to 300 nm enabling observation of worms in the cultivation chamber. As
depicted in Figure 1, the air vent channels further facilitate gas exchanges between the
cultivation chamber and the environment. Each cultivation chamber can accommodate about
1,000 worms from three generations. Based on the criteria of growth, reproduction and behavior,
worms grown in the CD are indistinguishable from those under standard laboratory conditions
(KIM et al. 2007).
The spin-stand system was originally designed for NASA to apply centrifugal force producing a
1G control in spaceflight. In the current study, this feature was used to generate hypergravity.
Rotation of the CD on the spin-stand applies a force on the liquids in the CD. This “centrifugal
force” can be precisely programmed and is identical for every cultivation chamber in the CD,
allowing worms with different genetic backgrounds and drug treatments to be assayed in parallel.
The force of 100G requires a rotation speed of 1,726 rpm. At 100G, inwards- and outwards-flow
of liquids in the cultivation chamber reach equilibrium within seconds, always leaving 70 ml of
culture medium in the cultivation chambers (Figure 1). Because of the gravity force, worms are
retained in the culture medium. The temperature in the cultivation chambers was measured
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using an EXTECH 470 Infrared Thermometer. It measures temperature utilizing a non-contact,
infrared thermometer and includes a built-in laser beam to target area. The thermometer has 8:1
distance to target ratio, 0.95 fixed emissivity. Based on three independent trials, each measured
five time points, 2 min for each time point, the temperature in the cultivation chambers was
maintained between 23 – 24oC.
In the current study, food was added to the culture medium in the cultivation chambers.
To prepare the food, a single colony of E. coli OP50 was inoculated into 250 ml of LB medium
shaking overnight at 37oC. The resulting bacteria were concentrated by centrifugation and the
pellet was re-suspended at the final concentration of 0.3g/ml of S-basal. 20 µl of the bacterial
suspension were added to 150 µl of S-medium in each cultivation chamber. For each
experiment, about 15 one-day old young adult animals were transferred into each cultivation
chamber. The 1G controls are the same strain raised in parallel, transferred to the CD cultivation
chambers, and rocked slowly on a rotator. All the experiments were performed at room
temperature (~22oC). For drug experiments, the drugs were dissolved in water and the resulting
solution was added to the medium to reach a final concentration of serotonin at 5 mg/ml and
fluoxetine at 0.5 mg/ml.
To monitor food consumption in different gravitational environmental conditions, E. coli
H115 DE3 expressing a red fluorescent protein (RFP) construct, pRSETB-mRFP, was used as a
food source. The RFP-E. coli was prepared the same way used for OP50, except that ampicillin
was added at a final concentration of 50 mg/ml LB medium to maintain the RFP plasmid.
Worms were raised with OP50 as the food source, transferred to CD cultivation chambers
containing RFP-E.coli, exposed to 100G or 1G for 15’ and 30’, and red fluorescence in the
worms was visualized using a Texas red filter. Worms fed with RFP-E. coli on plates at 1G
were observed as an additional control.
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To test DAF-16::GFP response to 100G in conventional test tubes, 8 –12 one-day old
adult animals were transferred to a 1.5-ml flat-bottom centrifugation tube (FastPlasmid‚ Kit,
Eppendorf) containing 150ml S-medium and 20ml of the bacterial suspension. The tubes were
transferred to a fixed-angle rotor in an Eppendorf centrifuge (5810R), and spun at 1,170 rpm
(100G) for 3 hr at the temperature of ~ 22oC.
Fluorescence Microscopy
All GFP reporters used in this study are integrated into the chromosomes. The expression of GFP
was observed using the Zeiss Axioplan II microscope and the Nikon Eclipse microscope,
equipped with a fluorescence light source. The images were captured with a Zeiss AxioCam
digital camera. To observe DAF-16 subcellular distribution in different genetic backgrounds, a
stably integrated DAF-16::GFP, zIs356 (HENDERSON and JOHNSON 2001) was crossed into
individual genetic backgrounds. To observe GFP subcellular distribution, 8-10 animals were
mounted to an agar pad containing 20mM sodium azide, and analyzed immediately (less than 10
min). Well-fed WT animals cultured under standard laboratory conditions were always assayed
in parallel. For quantification of DAF-16::GFP nuclear accumulation, animals were classified
into three categories as depicted in Figure 2a.
To test DAF-16::GFP response to heat shock, L4 animals were transferred to NGM plates
seeded with OP50 food, and incubated at 20oC for 24 hr. The plates were shifted to 35oC for 2
hr, and the subcellular localization of DAF-16::GFP in the animals was observed.
To quantify GFP intensity of tph-1::gfp and daf-7::gfp in individual neurons, GFP images
were captured using a 40x lens at a fixed exposure time, and the fluorescence over a 25x25 pixel
area within a neuron was quantified, using the Adobe Photoshop 6.0 software.
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To observe the effect of aldicarb on body wall muscle sarcomeres, worms carrying a
MYO-3::GFP transgene were incubated in the medium containing 1mM aldicarb for 20 minutes,
and the GFP patterns were observed.
Behavioral assays
Touch avoidance response was assayed by touches at the shoulder and tail regions of individual
worms. In general, worms move backward in response to a gentle touch at the head region, and
move forward when they are touched at the tail (CHALFIE and SULSTON 1981; SZE et al. 1997).
Immediately following 3 hr exposure to 100G, individual worms were touched with a thin
platinum wire 10 times alternately at the shoulder and tail regions, and the number of responses
was scored. Such platinum wire touches have the potential to activate the touch receptor
neurons and the PVD neurons (GOODMAN AND SCHWARZ, 2003; O’HAGAN AND CHALFIE, 2006)
Odor attraction assays were performed according to a standard protocol (BARGMANN et al.
1993). The dilution of the odorant diacetyl in ethanol was 1:1000. The odortaxis index was
calculated as [(number of worms at attractant)-(number of worms at solvent ethanol)]/(total
number of worms moved).
Statistics
Routine statistical analyses were performed using Minitab 12.1 (Minitab Inc., 1998). For
comparisons between more than two groups an ANOVA (one-way) was used. When testing
between more than two groups treated in two different ways a General Linear Model (GLM) was
used. This was followed by a Tukey's pair-wise multi comparison procedure. For comparisons
between two test groups a student’s t-test was carried out.
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Fat storage assay
We used Sudan black staining to visualize the fat storage in worms, using the Ogg-Ruvkun
protocol (OGG et al. 1997). Briefly, WT worms were exposed to 100G for 3 hr or 12 hr, fixed
immediately in 1% paraformaldehyde in phosphate-buffered saline, frozen at -70°C, thawed,
washed, and then incubated overnight in saturated Sudan black solution.
RESULTS
The mechanical stress of hypergravity induces DAF-16 nuclear accumulation
We have devised a compact disc (CD) based microfluidic platform to enable the study of
genetic, cellular, physiological parameters in living C. elegans as a function of gravitation level
(Figure 1). This CD system was designed for use in space as a 1G control to assess factors such
as microgravity, radiation, and vibration on C. elegans behavior and gene expression (KIM et al.
2007). In this system, worms are maintained in the cultivation chambers embedded in the CD,
and the CD is fixed onto a spin-stand system. By programming rotational speed, a precise
gravitational force is delivered to the cultivation chambers. Prior studies have established that
worms cultivated in the CD exhibit the tempo of development, rate of population growth and
brood size as they grow under standard laboratory conditions (KIM et al. 2007). In this study,
we used this CD cultivation system to test the effect of hypergravity on various genetic and
cellular parameters in C. elegans.
Hypergravity paradigms have been used to complement studies on microgravity and as an
experimental approach towards understanding the role of mechanical stress in biological systems
(LE BOURG 1999; MARKIN et al. 2004; MOREY-HOLTON 2003; OKAICHI et al. 2004; SOGA et al.
2005; TOU et al. 2002). The ability of a living system to withstand increased gravitational force
is related to size. For example, rats can survive up to 15G, whereas single cells and nematodes
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can withstand 100,000G (MOREY-HOLTON 2003). 100G is well within the range of centrifugation
force commonly used in the laboratory for collecting living C. elegans from a solution or a liquid
culture for subsequent behavioral assays and does not cause any significant detrimental effect on
worms. We, therefore, used 100G as a hypergravity paradigm.
Because FoxO transcription factors regulate homeostatic stress response pathways in
diverse organisms, and because the subcellular distribution of the FoxO factor DAF-16 is known
to be modulated by the environment and can be observed in living worms, we used DAF-16 as a
tool to monitor physiological response to gravity. We compared the subcellular distribution of
the DAF-16 protein tagged to a green fluorescent protein (DAF-16::GFP) in living worms
exposed to 1G and 100G (Figure 2a). As in worms raised under standard laboratory culture
conditions ( OGG et al. 1997; HENDERSON and JOHNSON 2001; LEE et al. 2001; LIN et al. 2001),
animals cultivated in the CD at 1G expressed DAF-16::GFP in most cell types including
muscles, intestine, hypodermis and many neurons, and GFP expression appeared to be diffuse in
the cytoplasm and nuclei. At the setting of 100G and over the course of 3 hr, worms retained
their morphological integrity. However, they displayed progressive DAF-16::GFP nuclear
accumulation in neuronal and non-neuronal cells throughout the body. DAF-16::GFP nuclear
accumulation became evident after 1 hr, and was further enhanced after 3 hr of 100G exposure.
This 100G-induced DAF-16 nuclear translocation was reversed after the animals returned to 1G
(Figure 2b) . These observations revealed that the FoxO pathway responds to the mechanical
loading of hypergravity.
Mechanical stress induces changes in metabolic profiles
In response to aversive environment, C. elegans modifies its metabolic profiles. For
example, under high growth temperature and starvation worms alter the activities of key
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metabolic enzymes, resulting in a shift of metabolic profiles favoring fat deposition, and this
metabolic shift is in part regulated by the DAF-2 insulin/IGF-1 receptor signaling to DAF-16
(OGG et al. 1997; ASHRAFI et al. 2003). It has been shown in rats that spaceflight causes changes
in insulin and glucose levels and alters the activity of enzymes involved in lipolysis (MACHO et
al. 2001). Also, hypergravity can cause weight increase and fat depositions (BOUET et al. 2004;
MARKIN et al. 2004; SMITH 1976). We therefore used fat storage as an assay to test the effect of
hypergravity on metabolism. Worms exposed to 100G for 12 hr showed increased fat content in
the intestine and hypodermis, as detected by Sudan black staining (Figure 2c). Increased fat
storage became noticeable in worms exposed to 100G for 3 hr, although the differences were
small (results not shown).
Hypergravity does not disturb feeding
An implicit concern with worms exposed to 100G was that the physical loading might
inhibit feeding, resulting in secondary changes in DAF-16 subcellular distribution and
metabolism. We addressed this question by monitoring food ingestion in worms exposed to
100G. We fed worms with E. coli cells expressing a red fluorescent protein (RFP), and
monitored the efficiency of red fluorescence to replace the colorless OP50 ingested prior to the
experiment (Figure 3). Within 15 minutes red fluorescence can be detected in the intestine, and
the intensity further increased after 30 minutes. There was no evident difference in the intensity
of red fluorescence between worms exposed to 100G and the ones kept at 1G throughout the
course of the experiment. These observations suggest that the function of the pharyngeal
muscles was preserved, and 100G did not significantly disturb feeding.
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Hypergravity induces muscular relaxation but does not disturb the integrity
To examine further the effects of hypergravity on the muscular system, we used GFP-
tagged myosin heavy chain protein MYO-3 (MYO-3::GFP) as a reporter to examine the body
wall muscles in animals exposed to 100G. There was no detectable difference in the
organization and morphology of the muscle fibers between animals exposed to 100G for 3 hr and
those at 1G. However, the muscle sarcomeres in worms exposed to 100G for 24 hr were
elongated and the muscle fibers were more stretched apart compared to 1G controls (Figure 4ab).
Unlike the acetylcholinesterase inhibitor aldicarb that causes body wall muscle hypercontraction
(MAHONEY et al. 2006) and irregular sarcomere organization (Figure 4c), the muscle fibers in
animals exposed to 100G for 24 hr remained evenly organized in parallel rows (Figure 4b),
demonstrating that the hypergravity force did not damage the structure of the body wall muscles.
The sarcomere elongation is likely a result of muscular adaptation to the chronic mechanical
loading imposed on the animal by the hypergravity force. It is noteworthy that this phenotype is
distinctly opposite to muscle cell shrinkage, shortening of muscle fibers and sarcomeres, and
muscle mass loss observed in rodents and humans under microgravity conditions (IKEMOTO et al.
2001; OHIRA et al. 2004; VANDENBURGH et al. 1999).
Neuronal integrity is preserved in animals exposed to hypergravity
We next tested the possibility that the physical force of 100G impairs neuronal functions
that in turn affect physiology. In C. elegans the integrity of chemosensory neurons influences
DAF-16 subcellular distribution, metabolism and ageing (ALCEDO and KENYON 2004; APFELD
and KENYON 1999). The microtubule-based cilia at the dendritic tip of the amphid chemosensory
neurons in the head are exposed to the external environment and express receptors and channels
that sense chemical and physical stimuli including odorants, soluble chemicals, food,
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temperature, osmotic strength, and mechanical touch at the nose (WARD et al. 1975, BARGMANN
and MORI 1997). Damaging the sensory structure of these neurons causes sensory deficits
(PERKINS et al. 1986) and DAF-16 nuclear accumulation (LIN et al. 2001).
We used GFP reporters to assess the effects of 100G on the structure of these
chemosensory neurons. osm-6 is expressed in all the chemosensory neurons (COLLET et al.
1998); 3 hr exposure to 100G did not produce a noticeable change in osm-6::gfp expression (data
not shown). We further examined the expression of specific genes in chemosensory neurons that
are known to regulate DAF-16. The ADF chemosensory neurons are a pair of serotonergic
neurons, and the pair of the ASI chemosensory neurons produces DAF-7/TGF-beta signal.
Reduction of daf-7 expression in ASI (LIN et al. 2001) or reduction in ADF of the tph-1 gene
that encodes the serotonin-synthesizing enzyme tryptophan hydroxylase (LIANG et al. 2006)
causes DAF-16::GFP nuclear accumulation. Over the course of 3 hr, exposure to 100G did not
cause a significant reduction of tph-1::gfp or daf-7::gfp expression in WT worms (Figure 5ab).
Prior studies showed that this daf-7::gfp reporter is significantly downregulated under the
conditions of starvation or high growth temperature (REN et al. 1996; SCHACKWITZ et al. 1996).
The normal daf-7::gfp expression level in animals exposed to 100G is another indication that
100G did not significantly alter growth temperature or disrupt feeding. Judged at the level of
fluorescence microscopy, there was no detectable change in the cell position or dendritic
morphology of ADF and ASI (Figure 5ab).
We also conducted behavioral assays to assess the function of the chemosensory neurons.
The activity of the AWA olfactory sensory neurons can influence DAF-16 (ALCEDO and
KENYON 2004). AWA senses the attractive odorant diacetyl (BARGMANN et al. 1993).
Dysfunction of the AWA neurons, their postsynaptic targets, or muscular function prevents
animals from responding to diacetyl. We used diacetyl sensation to test whether the physical
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force of 100G damages AWA. Worms assayed immediately following 3 hr exposure to 100G
responded to diacetyl indistinguishably from their siblings kept under 1G (Figure 5c). Together,
these results indicate that within this time frame the gross neuronal structure, the function of the
amphid chemosensory neurons, and neuromuscular networking in C. elegans were preserved
under 100G. Therefore, DAF-16 nuclear accumulation in animals exposed to 100G is unlikely to
be a consequence of chemosensory damage.
The MEC-10 DEG/ENaC channel of touch receptor neurons couples hypergravity and
DAF-16
Having established that the hypergravity induced DAF-16 nuclear accumulation
independently of starvation or neuronal and muscular damage, we sought for molecular
mechanisms underlying gravity response. In vertebrates, gravity is sensed by three classes of
receptor cells: hair cells of the vestibular system, proprioceptors located in the muscle joints, and
mechanoreceptors located in various regions of the body. Mechanoreceptors are among the most
conserved signaling components across phyla. In animals, mechanoreceptors are generally
located in the epidermis and are covered by a connective tissue capsule. Stimuli, including
touch, stretch, vibration and pressure, deform mechanoreceptors or displace the attachment
between ion channels of the mechanoreceptors and the extracellular matrix; this alters the
probability of the channel opening and triggers signaling cascades to generate behavioral and
physiological responses (SUKHAREV and COREY 2004). In C. elegans, a set of six touch receptor
neurons senses mechanical stimuli along the body (CHALFIE and AU 1989) (Figure 6a). Like
mammalian mechanoreceptors, the processes of these touch receptor neurons are closely attached
to the body wall, packed with specialized microtubules, and exhibit prominent extracellular
matrix (CHALFIE et al. 1985). In addition to connections to the locomotory circuitry, these
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neurons also have synapses connecting to many cells that are not directly involved in movement
(CHALFIE et al. 1985), suggesting that sensory perception of mechanical stimuli by these neurons
could have a broad impact on the animal. We first asked whether 100G damages these touch
receptor neurons. We used mec-4::gfp (DRISCOLL and CHALFIE 1991; HUANG and CHALFIE
1994) to visualize the touch receptor neurons in WT animals exposed to 100G and 1G. The
morphology of the cell body and the processes of touch receptor neurons in animals exposed to
100G for 3 hr were indistinguishable from those seen in animals at 1G (Figure 6c). Assayed
immediately following 3 hr exposure to 100G, worms responded instinctively to touches applied
to anterior and posterior regions of the body (Figure 6b). Thus, 100G forces did not undermine
the ability of the touch receptor neurons to sense and transmit sensory information.
We next tested a hypothesis that these touch receptor neurons function to translate the
mechanical stress of 100G into a biological signal inducing DAF-16 nuclear accumulation.
Mechanosensation of these neurons involves a membrane protein complex of four gene products.
The mec-4 and mec-10 genes encode DEG/ENaC proteins that form the pore of the
mechanosensory channel and are expressed in the plasma membrane of the touch receptor
neurons (DRISCOLL AND CHALFIE 1991; HUANG AND CHALFIE 1994; GOODMAN AND SCHWARZ,
2003; O'HAGAN AND CHALFIE, 2006). The transmembrane paraoxonase-like protein MEC-6
interacts with MEC-4, localizing the mechanosensory channel to puncta clusters along the axon
of the touch receptors (CHELUR et al. 2002). The extracellular protein MEC-9 is secreted by the
touch receptor neurons to provide an extracellular attachment point for the channel complex (DU
et al. 1996). Signaling through the mechanosensory channel requires MEC-7, a b-tubulin that
produces touch receptor-specific 15-protofilament microtubules (SAVAGE et al. 1989).
Dysfunction of any of these components causes worms to be insensitive to gentle touch along the
body. To investigate the possibility of the DEG/ENaC channel as a primary transducer of
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gravity, we tested whether disruption of the channel function could decouple the physical force
and DAF-16 subcellular distribution. We crossed the same DAF-16::GFP transgene into worms
bearing a loss-of-function mutation either in mec-4, mec-6, mec-7, mec-9, or mec-10, and
observed GFP subcellular distribution in the mutant animals exposed to 100G and 1G. At 1G,
there was no appreciable difference in DAF-16::GFP subcellular distribution between WT and
the Mec mutants. However, compared to WT animals assayed in parallel, each of the mec- gene
mutations attenuated or blocked 100G-induced DAF-16::GFP nuclear accumulation (Figure 7a).
Interestingly, two mec-4 alleles both conferred less inhibition of DAF-16::GFP nuclear
accumulation than the other Mec mutants. While all these mec- genes are expressed in the six
touch receptor neurons, mec-6, mec-7, mec-9 and mec-10, but not mec-4, are additionally
expressed in the pair of PVD neurons. It has been proposed that PVD has the characteristics of
the Drosophila multidendritic sensory neurons that mediate proprioceptive and nociceptive
functions (O’HAGAN AND CHALFIE, 2006), and has been shown to sense harsh mechanical
stimuli (WAY AND CHALFIE, 1989). It is possible that the physical force of 100G activates the
touch receptors as well as PVD to promote DAF-16 nuclear accumulation.
To ensure that the DEG/ENaC channel complex indeed is required for DAF-16 response
to the mechanical stress of hypergravity, we reproduced the same experiment in a more
conventional laboratory setting. We placed worms in flat bottom culture tubes and spun the
worms in a conventional centrifuge at the level of 100G. More than 60% of WT worms showed
DAF-16::GFP predominantly in the nuclei, whereas less than 10% of mec-9 and mec-10 mutants
showed strong DAF-16::GFP nuclear accumulation after 3 hr of chronic centrifugation (Figure
7b). The results confirmed our work with the CD. Both WT and Mec mutants incubated in the
culture tubes for 3 hr at 1G or 100G showed a slight increase in DAF-16::GFP nuclear
accumulation relative to their siblings incubated in the CD chambers (Figure 7ab). One plausible
Kim et al. Page 19 8/6/07
explanation for the quantitative difference in the results could be inadequate oxygen in the
conventional cultural tubes spun in a closed centrifuge, whereas the microchannels, air vent
channels and the open-air spin-stand provide a better ventilation system for the cultivation
chambers in the CD. The results from both CD and the conventional lab setting suggest that
signaling of the MEC-4/MEC-10 mechanosensory channel influences DAF-16 response to
hypergravity.
In WT worms DAF-16 nuclear accumulation can be induced by another physical stress
–– heat (HENDERSON and JOHNSON 2001; LIN et al. 2001). The temperature in the cultivation
chambers under 100G was between 23 - 24oC. Because the optimal C. elegans growth
temperature is 20oC, we first tested the possibility of DAF-16::GFP nuclear accumulation in WT
being a result of the temperature elevation during the 100G treatment. There was no detectable
DAF-16::GFP nuclear accumulation in WT animals incubated on NGM at 1G, 24oC or 26oC, for
3 hr (three independent trials, 20 animals/temperature/trial). Thus, within this time frame mild
heat stress alone would not be sufficient to induce DAF-16::GFP accumulation.
We next tested whether defective DEG/ENaC signaling specifically decouples
mechanical stress and DAF-16, or the mutations also obstruct DAF-16 response to other stress
physical signals. We exposed well-fed WT and mec-6, mec-9 and mec-10 mutant animals to
35oC heat shock, and observed DAF-16::GFP subcellular distribution. After exposure to the heat
for 2 hr, both WT and Mec mutant animals showed strong DAF-16::GFP nuclear accumulation
(Figure 7c). The percentage of mec-9 mutant animals exhibiting predominant DAF-16::GFP
nuclear accumulation appears to be slightly lower than that seen in the other strains, but the
difference is not significant (P=0.097, Student’s t-test). These observations indicate that
mechanical stress of hypergravity and thermal stress are sensed by distinctly different receptors
to independently regulate DAF-16 activity. Our results are consistent with the model that the
Kim et al. Page 20 8/6/07
MEC-4/MEC-10 channel specifically regulates DAF-16 response to mechanical force.
However, we do not exclude the possibility that 100G could produce some minor side effects
that also promote DAF-16::GFP nuclear translocation.
Drugs that enhance serotonin signaling confer resistance to mechanical stress
The serotonergic system functions as a neuromodulator to control neuronal plasticity and
physiological adaptation in both vertebrates and invertebrates (AZMITIA 1999; CHAOULOFF et al.
1999). Drugs that target the serotonergic system are effective in the treatment of a wide variety
of stress symptoms (LUCKI 1998). Other work in our laboratory established that fluctuation of
serotonin signaling modulates DAF-16 subcellular localization (LIANG et al. 2006). As the first
step towards identification of drugs that might be able to modify physiological response to
changes in gravity, we tested whether an excess of serotonin can suppress hypergravity-induced
DAF-16 nuclear accumulation. Applying serotonin or the selective serotonin reuptake inhibitor
(SSRI) fluoxetine to WT worms during their exposure to 100G significantly attenuated DAF-
16::GFP nuclear accumulation compared to their untreated siblings (Figure 2d), showing that
drugs that enhance serotonin signaling can mitigate the response elicited by the mechanical stress
of hypergravity.
DISCUSSION
This paper describes a paradigm for a systematic genetic, cellular, and physiological
survey of the effects of an ambient physical force on living animals undergoing complex
behavior. While a comprehensive appreciation of the effects of gravity on biology requires
investigations into multiple generations under various levels of hypogravity and hypergravity,
the ability of C. elegans to withstand the mechanical stress of 100G affords an opportunity to
Kim et al. Page 21 8/6/07
investigate molecular pathways that transduce mechanical forces into biological regulators and to
identify genetic targets of mechanical stress in the context of a whole animal.
Our genetic analysis of mechanosensory mutants revealed a molecular mechanism by
which a physical cue regulates metabolism and physiology. The phenotype of Mec mutants
corroborates prior studies of chemosensory mutants (LIN et al. 2001), both suggesting that the
perception of the environment regulates DAF-16, thereby influencing physiology. However,
there is a major distinction between the roles of chemosensation and mechanosensation. In the
case of chemosensation, mutants that cannot sense the chemical environment exhibit DAF-16
nuclear accumulation as WT animals exposed to aversive chemicals cues (LIN et al. 2001),
suggesting that inactivation of these chemosensory neuronal signaling produces a perception of
“stress” and that favorable chemical cues are necessary to suppress DAF-16 nuclear
translocation. By contrast, Mec mutants exposed to 100G exhibited the DAF-16 subcelluar
distribution as if they were under the earth gravity of 1G, implying that in the absence of MEC-
4/MEC-10 channel signaling mechanical loading cannot promote DAF-16 nuclear accumulation.
Importantly, Mec mutants that cannot sense hypergravity can vigorously respond to heat
stress. While both heat and gravity are physical cues, temperature is a major determinant of all
chemical action and interactions, whereas mechanical forces are generally considered lacking
chemical information. Our data demonstrate that the MEC-4/MEC-10 channel selectively senses
mechanical stress, and heat stress is sensed by an independent mechanism. The specific
receptors transducing heat and chemical cues have not yet been reported; further studies are
necessary to elucidate how individual sensory perceptions are integrated to modulate behavior
and physiology.
The touch- or stretch-sensitive channels are among the most ancient signaling molecules
conserved from bacterium to man (ANISHKIN and KUNG 2005; INGBER 2006; SUKHAREV and
Kim et al. Page 22 8/6/07
COREY 2004), and the FoxOs and its downstream stress response pathways reflect an
evolutionary conserved adaptive mechanism (ACCILI and ARDEN 2004). It has been shown in
plants that blockade of stretch-activated channels by drugs prevents hypergravity-induced growth
retardation (SOGA et al. 2005). Hypergravity also induces the expression of a battery of
homeostatic stress response genes that regulate growth, proliferation, antioxidant and cell death
in culture mammalian cells and humans (MARKIN et al. 2004; OKAICHI et al. 2004) and causes
lifespan extension in male Drosophila (LE BOURG 1999); all these phenotypes have been shown
to be regulated by FoxOs. It will be interesting to determine whether mechanosensory channel
activity also modulates FoxOs and their downstream pathways in Drosophila and mammals.
Our pharmacological experiments showed that serotonin and fluoxetine can partially
suppress DAF-16::GFP nuclear accumulation in WT animals exposed to 100G. The precise role
of serotonin in the touch receptor function is not clear. Serotonergic neurons are not connected
to the touch receptors (WHITE et al. 1986). Furthermore, applying serotonin and fluoxetine also
inhibits DAF-16 response to starvation (LIANG et al. 2006). One plausible explanation is that
serotonin signaling produces a more general effect of inhibition of DAF-16 nuclear accumulation
as a primarily antagonist of stress responses. The development of the C. elegans hypergravity
paradigm should enable the systematic analysis to identify genes and biochemical pathways that
sense and respond to mechanical stress and potential drugs that may modify them.
ACKNOWLEDGEMENT
We thank S. Sandmeyer for inspiring discussions, J. Hines and T. Ricco from NASA for their
contribution in technical insights, T. Stiernagle and the Caenorhabditis Genetics Center for worm
strains. We specially thank J. Gargus for providing the facility where some of the experiments
were carried out and his critical reading of the manuscript. This work was supported by grants
Kim et al. Page 23 8/6/07
from NASA (to M. Madou), a Marie Curie fellowship (to C. Dempsey) and NIHMH (to J. Sze).
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Figure legends
Figure 1. Computer Numeric Control (CNC) machined CD assembly used for C.elegans
hypergravity experiments
a. A photograph of the CD cultivation platform (12 cm in diameter and 4 mm thickness). Each
CD contains three identical cultivation units. The CD can be fixed onto a spin-stand system,
which is equipped with precise angular positioning and programmable rotations per minute
(RPM) (Kim et al., 2007). The rotation speed for 100G is 1,726 rpm.
b. A schematic illustration of the microfluidic structure of a C. elegans cultivation unit. It is
comprised of a nutrient chamber (1), a cultivation chamber (2), and a waste chamber (3). These
chambers are connected by microchannels shown in yellow. Also shown in yellow, each
chamber has a pair of air vent channels to facilitate gas exchanges. Notice that this microfluidic
structure was designed for space applications that normally operate at low rotation speed to
produce a 1G reference in spaceflight. In hypergravity experiments, food was directly added to
the cultivation chamber.
c. A schematic depiction of fluidics in the cultivation chamber under 100G. Because of the high
rotation speed, which overcomes the surface tension, liquid will flow into the waste chamber
until the liquid level drops below the microfluidic channels connected to the waste chamber.
Shown in blue, the equilibrium level has a volume of 70 ml. Among advantages of this CD over
conventional culture tubes are the wide front edge of the cultivation chamber that prevents
worms clumping together during high-speed centrifugation and the microchannels and air vent
channels that provide a better ventilation system for the cultivation chambers. The temperature
in the cultivation chambers under 100G was maintained between 23oC – 24oC.
Kim et al. Page 29 8/6/07
Figure 2. Mechanical stress of 100G induces DAF-16::GFP nuclear accumulation and
excess fat storage
a. DAF-16::GFP subcellular distribution in WT animals. Under 1G, DAF-16::GFP displayed a
diffuse pattern in neuronal and non-neuronal cells throughout the body. The GFP expression in
the hypodermis is shown. After 1 hr exposure to 100G, DAF-16::GFP was distinctly enriched in
the nuclei; the hypodermis and muscle cells are shown. After 3 hr exposure to 100G, DAF-
16::GFP was predominantly localized in the nuclei. We classified DAF-16::GFP subcellular
distribution into 3 categories: not localized, as exhibited by the animal under 1G control shown
in the top photomicrograph; partial nuclear accumulation, as shown in the two middle
photomicrographs; and predominant nuclear accumulation, as shown in the two bottom
photomicrographs. This experiment was repeated by three people, including a blind test for
animals exposed 1G and 100G.
b. Kinetics of DAF-16::GFP nuclear accumulation in worms exposed to 100G and recovery
after they returned to 1G. Each bar represents the mean of at least three independent trials, 15-20
animals per trial.
c. Hypergravity causes increased fat storage. Fat content in animals exposed to 1G and 100G
was detected by Sudan black staining. Three independent experiments were carried out
including a blind test for animals exposed to 1G and 100G, 40 - 45 animals/treatment/trial. More
than 50% of animals exposed to 100G for 12 hr showed increased fat accumulation based on
visual inspection, and the photomicrographs show the representative staining patterns.
Kim et al. Page 30 8/6/07
d. Exogenous serotonin and fluoxetine suppress 100G-induced DAF-16::GFP nuclear
accumulation. WT animals were exposed to 100G in the presence or absence of a drug
treatment, and the percentage of animals that exhibited predominant DAF-16::GFP nuclear
accumulation was scored. Each bar represents three independent trials ± SEM, each in
triplicates. 68 – 70 animals were assayed for each treatment.
Figure 3. Feeding behavior is preserved in worms exposed to 100G.
Representative photomicrographs showing worms fed with E. Coli expressing RFP at 1G and
100G. a, c, e and g show RFP visualized by a Texas red filter, and b, d, f and h show
superposition of RFP and auto-fluorescence of the gut visualized by a FITC filter. These
observations are based on 6 – 9 independent experiments, with 100G and 1G conditions tested in
parallel, at least 10 worms/trial/gravity condition. All the animals shown are young adults, and
the anterior is toward the left.
Figure 4. The muscular structure is preserved in worms exposed 100G.
Body wall muscle sacromers in living animals were observed using GFP-tagged myosin heavy
chain protein MYO-3 (MYO-3::GFP). One-day old young adult animals were exposed to 100G
for 3 hr or 24 hr, and examined immediately afterwards. Exposure to 100G for 3 hr did not
produce a detectable change (not shown). After 24 hr exposure, although the sarcomeres were
longer relative to the 1G controls (outlined by dashes), the muscle fibers remained well
organized. By contrast, age-matched animals under 1G treated with the cholinesterase inhibitor
aldicarb, which causes muscle hypercontractions, displayed densely packed muscle fibers, and
the muscle cells were shrunken and crenated, an antithesis of the stretched parallel striation seen
Kim et al. Page 31 8/6/07
in worms under 100G. These observations are based on three independent experiments, with at
least 10 worms/trial/condition.
Figure 5. The integrity of the nervous system is preserved in worms at 100G for 3 hr.
a. Serotonergic neurons in the head region were visualized by a GFP reporter of the gene
encoding the serotonin-synthesizing enzyme tryptophan hydroxylase (tph-1::gfp). Over the
course of 3 hr, GFP levels were not reduced in the ADF chemosensory neurons or the NSM
secretory neurons (p>0.05, one-way ANOVA).
b. The ASI chemosensory neurons were visualized by a GFP reporter of the daf-7/TGF-beta
gene (daf-7::gfp). Unlike under starvation and high growth temperature (Ren et al., 1996;
Schakwitz et al., 1996), the expression level of daf-7::gfp was not significantly reduced in
worms under 100G (p> 0.05, one-way ANOVA). Each bar represents the mean±SEM of at least
three trials. All animals assayed were one-day old young adults. n = number of animals in
which GFP was quantified. AU: arbitrary units.
c. Olfactory sensation to the attractant diacetyl. Worms were exposed to 100G for 3 hr, and
assayed immediately afterwards. There was no significant difference in the olfactory sensitivity
between worms exposed to 100G and the 1G controls (p> 0.05, Student’s t-test). Each bar
represents the mean±SEM of at least three trials, 24 - 30 animals/gravity condition/trial. One-
day old young adults were assayed.
Figure 6. Structure and function of the touch receptor neurons after 3 hr exposure to 100G
Kim et al. Page 32 8/6/07
a. A schematic representation of the touch receptor neurons. Adapted from Chalfie and Au
(1989).
b. Touch response. The animals were assayed within 20 minutes after the return from 100G.
Individual worms were touched with a thin platinum wire 10 times alternately at the shoulder
and tail regions, and the number of responses was scored. Each bar represents the mean±SEM of
at least two trials, 10 animals per trial. All animals assayed were one-day old young adults.
While the six touch receptor neurons are required to respond to touches at the shoulder and tail
regions, platinum wire touch could additionally activate the PVD neurons (WAY AND CHALFIE,
1989)
c. The cell body and processes of touch receptor neurons visualized by mec-4::gfp. The
worms were exposed to 100G for 3 hr. The left photomicrograph shows the image of an entire
animal at x5 magnification, and the right photomicrograph shows the ALM process extending to
the head region at x20 magnification. The anterior is toward the left.
Figure 7. Mutations in the MEC-4/MEC-10 DEG/ENaC mechanosensory channel signaling
block 100G-induced DAF-16 nuclear accumulation
a. – b. DAF-16::GFP subcellular distribution in worms exposed to 100G in the CD platform (a)
and a conventional centrifuge (b) for 3 hr. One-day old young adults were assayed. WT and
Mec mutants were always tested in parallel. Each bar represents the mean±SEM of at least three
trials, 10 – 12 animals/genotype/trial, including a blind assay for genotypes. There is a
significance difference (p<0.0001, GLM) found between the percentage of WT displaying
predominant DAF-16::GFP nuclear accumulation and every of the Mec mutant strains examined
Kim et al. Page 33 8/6/07
in both hypergravity paradigms. However, mec-4 mutants showed higher frequencies of partial
DAF-16::GFP nuclear accumulation.
c. Mechanosensory mutations do not affect DAF-16 response to heat stress. Like WT
worms, mec-6, mec-9, and mec-10 mutants exposed to 35oC for 2 hr exhibited DAF-16::GFP
nuclear accumulation. Each bar represents three independent experiments, each with 15
worms/condition. The frequency of mec-9 animals showing predominant DAF-16::GFP nuclear
accumulation appears to be lower than that of the other strains, but the difference between mec-9
and WT is not significant (P=0.097, Student’s t-test).
The classification of DAF-16::GFP nuclear accumulation is described in Figure 2.
Kim et al. Page 34 8/6/07
1
2
3
a. b.
Kim et al. Figure 1
2
c.
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Kim et al. Figure 4
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. a. tph-1::gfp expression
Gravitational environment1G 100G
20
40
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ex
c. Olfactory sensation
Kim et al. Figure 5
b. daf-7::gfp expression
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GF
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60
100
140
0 1 2 3100G exposure (hr)
n=20 n=14 n=19 n=19 n=36 n=23 n=21 n=39
Kim et al. Page 39 8/6/07
.a. Touch receptor neurons
Fre
qu
ency
of
resp
on
ses
(%)
Gravitational environment1G 100G
20
40
60
80
100
0
b. Mechanosensation
Kim et al., Figure 6
n=20n=29
c. mec-4::gfp (100G)
Kim et al. Page 40 8/6/07
.
a. DAF-16::GFP nuclear accumulation under 100G (CD)
b. DAF-16::GFP nuclear accumulation under 100G (tube)
Kim et al., Figure 7
Predominant nuclear Partial nuclear Not localized
WT mec-9 mec-10
1G 100G 1G 100G 1G 100G0
20
40
60
80
% A
nim
als
100
20oC
2hr
WT mec-9 mec-6 mec-10
20oC 20oC 20oC 20oC35oC 35oC 35oC 35oC2hr 2hr 2hr
0
20
40
80
100
% A
nim
als
60
c. DAF-16::GFP nuclear accumulation under heat stress
1G 100GWT
1G 100Gmec-6
1G 100Gmec-9
1G 100Gmec-10
1G 100Gmec-4
(e1611)
1G 100Gmec-4(u253)
1G 100Gmec-7
0
20
40
60
80
% A
nim
als
100