gravity force transduced by the mec-4/mec-10 …...2007/08/24 · nahui kima,b, catherine m....

Kim et al. 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. 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. 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. 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

Kim et al. 8/6/07

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.

Kim et al. 8/6/07

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:

Kim et al. 8/6/07

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

Kim et al. 8/6/07

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.

Kim et al. 8/6/07

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.

Kim et al. 8/6/07

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.

Kim et al. 8/6/07

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.

Kim et al. 8/6/07

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 8/6/07

1

2

3

a. b.

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2

c.

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. a. tph-1::gfp expression

Gravitational environment1G 100G

20

40

60

80

100

0

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c. Olfactory sensation

Kim et al. Figure 5

b. daf-7::gfp expression

20

60

100

140

0 1 2 3

GF

P in

ten

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(A

U)

100G exposure (hr)

NSM

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AU

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n=20 n=14 n=19 n=19 n=36 n=23 n=21 n=39

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.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. 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

gravity force transduced by the mec-4/mec-10 …...2007/08/24 · nahui kima,b, catherine m....

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