 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 1999, 19(6):1952-1958
Distinct Signaling Pathways Mediate Touch and Osmosensory
Responses in a Polymodal Sensory Neuron
Anne C.
Hart1, 2,
Jamie
Kass3,
Jonathan E.
Shapiro2, and
Joshua M.
Kaplan4
1 Department of Pathology, Harvard Medical School, and
2 Massachusetts General Hospital Cancer Center,
Charlestown, Massachusetts 02129, 3 Department of Genetics,
Harvard Medical School, Department of Molecular Biology, Massachusetts
General Hospital, Boston, Massachusetts 02114, and
4 Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720-3200
 |
ABSTRACT |
The Caenorhabditis elegans ASH sensory neurons
mediate responses to nose touch, hyperosmolarity, and volatile
repellent chemicals. We show here that distinct signaling pathways
mediate the responses to touch and hyperosmolarity. ASH neurons
distinguish between these stimuli because habituation to nose touch has
no effect on the response to high osmolarity or volatile chemicals
(1-octanol). Mutations in osm-10 eliminate the response
to hyperosmolarity but have no effect on responses to nose touch or to
volatile repellents. OSM-10 is a novel cytosolic protein expressed in
ASH and three other classes of sensory neurons. Mutations in two other
osmosensory-defective genes, eos-1 and
eos-2, interact genetically with osm-10.
Our analysis suggests that nose touch sensitivity and osmosensation occur via distinct signaling pathways in ASH and that OSM-10 is required for osmosensory signaling.
Key words:
Caenorhabditis elegans; osmosensation; mechanosensation; neurodegeneration; OSM-10; eos-1; eos-2; degenerin
| |
INTRODUCTION |
A class of polymodal sensory neurons
has been described in the nematode Caenorhabditis elegans.
The bilateral ASH sensory neurons mediate the responses to nose touch,
hyperosmolarity, and volatile repellents; each stimulus evokes backward
locomotion (Bargmann et al., 1990 ; Kaplan and Horvitz, 1993; Troemel et
al., 1995). ASH neurons provide direct synaptic input to command
interneurons that drive locomotion (White et al., 1986). An AMPA-type
glutamate receptor, GLR-1, is expressed in ASH synaptic targets and is
required for ASH-mediated touch sensitivity but not for osmotic or
volatile sensitivities mediated by ASH (Hart et al., 1990; Maricq et
al., 1995). These results suggest that the ASH-to-interneuron synaptic signals evoked by touch differ from those signals evoked by osmotic and
volatile repellents. However, it is unclear whether common or distinct
pathways mediate sensory transduction by the different ASH sensory stimuli.
Touch and hyperosmolarity are both thought to be detected by
mechanically gated ion channels. The C. elegans degenerin
proteins have been proposed to act as mechanosensory receptors. The
degenerins MEC-4 and MEC-10, distantly related to epithelial sodium
channels, are required for the response to body touch mediated by the
"touch neurons" (ALM, PLM, AVM, and PVM). Dominant mec-4
and mec-10 mutations cause neurodegeneration, hence the name
degenerins. The putative mechanoreceptor complexes are proposed to
contain combinations of degenerin subunits and proteins that tether
these complexes to cytoskeletal and extracellular matrix proteins
(Driscoll and Kaplan, 1997). Although these complexes are an attractive
model for the mechanoreceptor of the touch cells, it is unclear whether other mechanosensory neurons like ASH use a similar receptor complex. An alternative candidate receptor is OSM-9, a C. elegans
homolog of the capsaicin receptor (Colbert et al., 1997). The
vertebrate capsaicin receptor encodes an ion channel gated by either
capsaicin or high temperature (Caterina et al., 1997). However,
osm-9 mutants are defective in response to all stimuli
detected by ASH (Colbert et al., 1997). No homologs of other receptors
implicated in osmosensation or mechanosensation in either yeast or
Escherichia coli have been identified in C. elegans.
Here we characterize further the mechanisms underlying ASH sensory
transduction and modality coding. We show that ASH neurons distinguish
between touch and other ASH stimuli and that ASH and the microtubule
touch cells use distinct mechanoreceptors, and we characterize a
modality-specific gene, osm-10 (osmosensory defective). A mutation in osm-10 disrupts ASH-mediated
osmosensation but has no effect on other ASH-mediated responses. Taken
together, our results suggest that the ASH responses to touch and
hyperosmolarity are mediated by distinct signaling pathways and
identify a novel intracellular protein that is required for osmosensation.
| |
MATERIALS AND METHODS |
Behavioral assays
Nose touch, osmotic avoidance, and volatile repellent assays
were performed as described previously (Culotti and Russell, 1978;
Kaplan and Horvitz, 1993; Hart et al., 1995). Behaviors were
quantitated as follows: nose touch avoidance (Not) was quantitated as
the percentage of trials in which animals responded to touch with an
eyelash by stopping forward movement or reversing, osmotic avoidance
(Osm) was quantitated as the percentage of animals that escape a ring
of 8 M glycerol in <8 min, and volatile avoidance (Sos)
was quantitated as the average time to initiate backward movement in
response to an eyelash dipped in 1-octanol. The morphology of ASH
neurons was examined by dye filling using DiD, DiI, or DiO (Molecular
Probes, Eugene, OR). DiD facilitates unambiguous identification of
green fluorescent protein (GFP)-expressing cells, even at low levels of
GFP expression (P. W. Faber, J. Alter, M. MacDonald, and A. C. Hart, unpublished observations). To generate transheterozygotes
unambiguously for behavioral assays, we used recessive genetic markers
(e.g., dpy-17) or osm-10:: GFP
reporter constructs to identify cross progeny. Strains used included
dpy-17 (e164) osm-10 (n1602); eos-1(nu288)
rtEx61(pKP#58); eos-1(nu288) lin-15(n765);
nDf16/dpy-17(e164) unc-32(e189); and individual mutant
strains listed in Tables 1-6. unc-8(n491n1193);
deg-1(u506u550) (HA27) was constructed from
dpy20(e1282ts)IV;deg-1(u506u550)X (HA4) and
unc-8(n491n1193). HA27 construction was confirmed by sequencing allele-specific polymorphisms. Animals were raised at 25°
unless otherwise indicated. Direct injection of pKP#58 into
eos-1 created rtEx61; strains were maintained by
selecting for GFP expression each generation on a GFP dissection
microscope (Leica/Kramer Scientific, Burlington, MA).
Positional cloning of osm-10
Mapping data. n1602 was mapped by
recombination with MT5427 (sma-3 6 mec-14 4 osm-10 7 ncl-1 9 unc-36) and
MT4837 (sma-3 8 osm-10 5 lin-39) to an
interval between mec-14 and lin-39 on chromosome III by the use of the Osm assay format described below. Cosmids from
the region were obtained from the C. elegans
genome-sequencing project. Twenty or more transgenic F3 generation
KP497 animals [osm-10 (n1602)III; lin-15 (n765) X] were
tested for osmotic avoidance for each cosmid or plasmid to assess
rescue activity. pHA#51 contains a 3.2 kb
PstI/XhoI fragment that corresponds to
12848-16050 of cosmid T20H4 and contains the 3' one-half of T20H4.2
and all of T20H4.1 and all of osm-10 except for part of the
3'-untranslated region.
Sequencing. The products of three independent PCR reactions
from osm-10(n1602) or eos-2(nu268) genomic DNA
were sequenced to identify allele-specific polymorphisms. No mutations
were identified in osm-10 exons in eos-2(nu268).
The genomic structure of osm-10 predicted by GENEFINDER and
the C. elegans genome-sequencing project was confirmed by
reverse transcription (RT)-PCR from wild-type animals using
Superscript Reverse Transcriptase (Life Technologies, Gaithersburg, MD)
and the Marathon cDNA Race Kit (Clontech, Palo Alto, CA). The
translation initiation site was confirmed by sequencing an RT-PCR
product containing a stop codon, in frame, 10 codons upstream of the
predicted initiator methionine codon.
osm-10(nr2076). The osm-10 deletion allele was
generated using a PCR-based strategy by the Nemapharm division of Axys
Pharmaceuticals (South San Francisco, CA) (Liu et al., unpublished observations).
osm-10 expression
Antisera. The second exon of osm-10 was
amplified by PCR and inserted into pET21b (Novagen, Madison, WI) for
bacterial expression with a polyHIS tag. Affinity-purified sera from
three immunized rabbits detected OSM-10 with varying specificity in
whole mounts fixed with Bowin's (Nonet et al., 1997) and on Western
blots. Confocal resolution was obtained with optical Z-series
sectioning and Openlab deconvolution image processing (Improvision,
Coventry, England). Western blots were blocked with 5% dry milk and
1% E. coli acetone powder in TBS plus Tween and were probed
with a 1:1000 dilution of affinity-purified (M. Koelle, personal
communication) anti-OSM-10 antisera. HRP-coupled goat anti-rabbit
antiserum (Amersham, Arlington Heights, IL) and ECL (Dupont NEN,
Boston, MA) were used to detect protein.
GFP constructs. In pKP#58 the GFP gene was
amplified from pPD95.67 and inserted in frame into the NruI site
[nucleotide (nt) 14155] in the osm-10 rescue construct
pKP#51 (see above). Additional GFP rescue constructs with different
insertion sites were generated; all resulted in the same expression
pattern in vivo. Coinjection of pKP#58 and pJM#24 into
lin-15(n765) and subsequent gamma irradiation resulted in
the insertion of nuIs11 into the X chromosome.
nuIs11 was backcrossed four times before laser ablation and
the other experiments reported in this manuscript.
Heterologous expression constructs. OSM-10 protein was
expressed using promoters from the srb-6 and
sre-1 genes using pHA#2 and pHA#1, respectively (Troemel et
al., 1995). srb-6 is expressed in ADL, ADF, ASH, PHA, PHB,
and the vulval region; sre-1 is expressed in ADL and
ASJ(faint). PCR-amplified osm-10 genomic DNA (T20H4, nt
13738-15801) replaced GFP in pTU#62 between
SmaI/EcoRI sites to create pKP#82.
SphI/SmaI sites in srb-6 and
sre-1 constructs (Troemel et al., 1995) were used to
subclone the srx promoter into pKP#82. Transgenic strain
construction was described in the previous section;
lin-15(n765) animals were injected.
Isolation of eos-2(nu268) and eos-1(nu288)
Hermaphrodite N2 animals were mutagenized with
ethylmethanesulfonate; 11,220 haploid genomes were screened, and 50 mutants that were defective in detecting high osmolarity but had normal dye filling of the amphid neurons were isolated, including
eos-2(nu268) and eos-1(nu288).
Osm assay. Approximately 20 µl of 8 M glycerol
(dyed with bromphenol blue) is distributed in a 1.5 cm ring on an agar
plate. The ring is allowed to dry for 2-3 min. Up to 300 well-fed
adult worms are washed three times with S Basal and placed in the
center of the ring. Excess S Basal is blotted off, and the percentage of animals escaping after 10 min is reported.
Mapping. eos-2(nu268) or eos-1(nu288)
was mapped with DA438 [bli-4(e397)I; rol-6(e187)II; daf-2(e1368)
vab-7(e1562)III; unc-31(e928)IV; dpy-11(e224)V; lon-2(e678)X].
Five of 17 eos-2(nu268) animals were heterozygous for
vab-7. eos-1(nu288) linkage to IV was confirmed with the dominant markers unc-8(n491dm) dpy-13(e184sd) IV.
Fourteen of 16 F2 animals that did not carry unc-8 dpy-13
were homozygous for eos-1(nu288).
| |
RESULTS |
Relationship between mechanoreceptors in ASH and microtubule
touch cells
Both touch and increased osmolarity are thought to be detected by
mechanically gated ion channels. The genes mec-2,
mec-4, mec-5, mec-6, mec-9,
and mec-10 encode components of a putative mechanoreceptor
complex that mediates touch sensitivity by the touch cells in C. elegans (Driscoll and Kaplan, 1997). These genes are not required
for ASH-mediated behavioral responses (Table 1). The degenerins DEG-1 and UNC-8 were
also considered likely candidates for ASH mechanoreceptors (Driscoll
and Chalfie, 1991; Huang and Chalfie, 1994; Driscoll and Kaplan, 1997)
because unc-8 is expressed in ASH (Tavernarakis et al.,
1997) and gain-of-function alleles deg-1(u38) and
deg-1(u506) cause ASH neurons to undergo neurodegeneration
(Table 2). The deg-1(u38)
mutation caused temperature-sensitive dominant nose touch
insensitivity, whereas deg-1 null alleles do not perturb the
nose touch response (Table 3)
(Garcia-Anoveros et al., 1995). Double mutants containing null alleles
for both unc-8 and deg-1 are normal for all
ASH-mediated responses (Table 3), suggesting that the mechanically
gated ion channels used by ASH for nose touch and osmotic detection may
be distinct from those used by the microtubule touch cells.
Animals distinguish between the ASH sensory stimuli
Although they mediate responses to three sensory stimuli, it is
possible that ASH neurons cannot distinguish between the different stimuli detected. If this were the case, one would predict that any
treatment that causes a decrement in one response would also produce a
defect in the other responses. We found that repeated nose touch
stimuli reduced responsiveness to further nose touch stimulation;
however, these habituated animals remained fully sensitive to
hyperosmolarity or volatile repellent chemicals (Table 4). Similar experiments habituating the
responses to hyperosmolarity or to volatile repellents were not
possible because chronic exposure to these stimuli is lethal. However,
a short exposure to 1-octanol partially habituates response without
deleterious effects; control animals reverse in 1.4 ± 0.3 sec,
but after two 15 sec exposures to 1-octanol, the average reversal time
is 4.9 ± 0.5 sec (n = 11 animals). This brief
habituation to 1-octanol neither increased nor decreased sensitivity to
nose touch (72 ± 6% response vs 80 ± 15% response in
control animals). These results suggest that the ASH neurons
distinguish nose touch from hyperosmolarity and the volatile repellent
1-octanol.
osm-10, a modality-specific gene required
for osmosensation
To identify genes involved in ASH sensory transduction, we sought
mutations that selectively impair ASH-mediated osmosensation but not
other ASH-mediated behaviors. One previously identified mutation,
osm-10(n1602), fit our criteria. As previously shown, osm-10(n1602) mutants failed to avoid either 8 M
glycerol or 4 M fructose (Table
5) (Bargmann et al., 1990), indicating
that they are defective for osmosensation. However, we found that
osm-10(n1602) animals responded normally to nose touch and
volatile repellents, indicating that the mutant ASH neurons retained
some sensory functions (Table 5). By contrast, other mutations that
cause generalized defects in ASH function or structure (e.g.,
osm-3 and eat-4) impaired multiple
ASH-mediated sensory behaviors (Table 1). The n1602 mutation
is likely to cause a severe defect in osm-10 function because the behavior of osm-10(n1602) homozygotes was
indistinguishable from that of osm-10(n1602)/nDf16
heterozygotes (Table 5).
We positionally cloned osm-10. We mapped
osm-10(n1602) to a small interval on chromosome III between
mec-14 and lin-39. A single cosmid clone from
this region, T20H4, and plasmid subclones derived from it were able to
rescue the osm-10 defect in transgenic animals (Fig.
1, top). In this manner, the
osm-10-rescuing activity was mapped to a single gene
predicted by the C. elegans genome-sequencing project
(T20H4.1), and the n1602 mutation was shown to correspond to
a missense mutation (E199K) in one of the predicted exons. We concluded
that the osm-10 gene corresponds to T20H4.1.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 1.
Positional cloning of osm-10.
Top, The osm-10 gene maps between
mec-14 and lin-39 on chromosome III. The
osmotic avoidance defect of osm-10(n1602) was rescued by
the cosmid T20H4 and the plasmid pKP#51 that contains the
osm-10 gene T20H4.1. See Results for details.
Bottom, The sequence of the osm-10 cDNA
was determined by RT-PCR, leading to the predicted protein sequence
shown. The n1602 mutation corresponds to the missense
mutation E199K; E199 is lowercase. Amino acids
translated in the predicted osm-10(nr2076) protein are
in the box. Potential S/T kinase and Y kinase sites are
indicated as follows (number of sites): protein kinase C (17) sites are
underlined; casein kinase II (11) sites are in
bold; cAMP- and cGMP-dependent kinase phosphorylation
sites (6) are in italics; and cyclin-dependent kinase
(5) sites are outlined.
|
|
After failing to isolate osm-10 cDNAs from available
libraries, we determined the intron/exon structure, the 5' and 3' ends of the osm-10 mRNA, by RT-PCR. These experiments confirmed
that the predicted OSM-10 protein contains 419 amino acids and is rich in serine and threonine residues (20.8%). Database searches for similar proteins failed to identify any osm-10 homologs.
OSM-10 contains 39 potential serine and threonine phosphorylation sites for members of the protein kinase C and the cAMP- and cGMP-dependent, casein, and cyclin-dependent kinase families. The n1602
mutation perturbs a potential tyrosine kinase phosphorylation site
(Fig. 1, bottom). An additional allele,
osm-10(nr2076), was obtained using reverse genetic
techniques and removed nucleotides 14324-15446 within T20H4.1. The
predicted mutant protein consists of 156 amino acids of OSM-10 before a
frame shift and premature termination of translation (after exon 2).
osm-10(nr2076) animals are phenotypically indistinguishable
from osm-10(n1602) animals.
Polyclonal anti-OSM-10 antisera detected a 49 kDa protein on Western
blots of wild-type and osm-10(n1602) animals (Fig.
2b), which is consistent with
the predicted size of the wild-type OSM-10 protein. Subcellular
localization of OSM-10 was addressed by expressing GFP reporter
constructs and by staining fixed animals with anti-OSM-10 antisera. We
detected OSM-10 expression in four classes of chemosensory neurons in
all larval and adult animals (ASH, ASI, PHA, and PHB). Expression
commences just before hatching. OSM-10 expression was never observed in
any other cells (Fig. 2a). The OSM-10 protein was uniformly
distributed throughout the cell bodies, sensory processes, and axons of
the expressing cells but was excluded from nuclei on the basis of
confocal resolution microscopy (data not shown). OSM-10 expression was
not altered in osm-10(n1602), osm-9(ky10), or
osm-3(p802) animals. OSM-10 expression was reduced to barely
detectable levels in osm-10(nr2076) animals. These results demonstrate that OSM-10 is a novel cytoplasmic protein expressed in the
osmosensory ASH neurons.
View larger version (38K):
[in this window]
[in a new window]
|
Figure 2.
a, OSM-10 is expressed in
ASH, ASI, PHA, and
PHB. Expression was monitored by staining fixed animals
in whole-mount preparations with a polyclonal rabbit anti-OSM-10
antibody. Cells were identified on the basis of their nuclear positions
and axon morphologies (Sulston et al., 1983; White et al., 1986). The
OSM-10 protein is diffusely distributed throughout the cell body,
sensory processes, cilium, and axons of expressing cells. The cellular
and subcellular expression pattern of the OSM-10:: GFP
reporter construct nuIs11I is identical. Cells in
gray are located on the right side of the
animal; cells in black are on the left.
Scale bars, 25 µm. b, Polyclonal rabbit anti-OSM-10
antisera recognize a 49 kDa band on Western blots from extracts of
wild-type, osm-10(n1602), osm-3(p802),
and nuIs11(pKP#58, pJM#24) animals. An additional band
at ~60 kDa corresponding to the OSM-10:: GFP fusion protein
is visible in the nuIs11I lane. The same blot probed
with anti-tubulin antibodies (N356; Amersham) is shown at the
bottom. c, OSM-10 immunoreactivity in
ASH neurons (visualized as in a) is
shown. OSM-10 immunoreactivity is excluded from the nucleus.
Left, Deconvoluted images of an
ASH cell body are shown. Middle,
Right, OSM-10 immunoreactivity is severely reduced in
osm-10(nr2076) animals but is found at wild-type levels
in eos-1(nu288) or eos-2(nu268) (data not
shown) animals.
|
|
The OSM-10 expression pattern suggested that ASI, PHA, and PHB might
also play a role in osmosensation. To test this possibility, we killed
ASH, ASI, PHB, and PHA (the primary synaptic input for PHB) with a
laser microbeam in various combinations, and the osmotic avoidance
behavior of the operated animals was analyzed (Table 6). We found that the osmotic avoidance
behavior of operated animals lacking ASH neurons (ASH ) was
indistinguishable from that of osm-3 mutants, whereas the
response of animals lacking ASI, PHA, and PHB was indistinguishable
from that of mock-ablated animals. osm-3 mutants have
defects in the sensory endings of amphid and phasmid neurons, including
ASH, ASI, PHA, and PHB (Perkins et al., 1986). On the basis of these
results, we conclude that the ASH neurons play the predominant role in
osmotic avoidance in this assay. It remains possible that ASI, PHA, and
PHB mediate osmosensory regulation of other behaviors.
The restricted expression of OSM-10, together with the osmotic
avoidance defects in osm-10 mutant animals, suggested that OSM-10 expression might be sufficient to confer osmotic sensitivity on
other sensory neurons. We tested this possibility by ectopically expressing OSM-10 using the promoter for the putative chemosensory receptor gene sre-1, which is expressed in the sensory
neurons ADL and ASJ (Troemel et al., 1995). Like ASH, the synaptic
targets of ADL include the interneurons that control locomotion in
C. elegans (White et al., 1986). Ectopic expression of
OSM-10 in ADL and ASJ did not rescue the osm-10(n1602)
mutant phenotype (81 ± 3% defective; n = 6 lines). In contrast, osmosensitivity was partially restored when OSM-10
was expressed in ASH, ADF, ADL, PHA, and PHB using the srb-6
promoter (44 ± 9% defective; n = 7 lines).
srb-6 is another putative chemosensory receptor gene
(Troemel et al., 1995). We conclude that OSM-10 expression is not
sufficient to confer osmosensitivity on other sensory neurons and that
OSM-10 expression in ASH, PHA, and PHB is sufficient for partial rescue
of osm-10.
Genes that interact with osm-10
We examined the role of OSM-10 in ASH-mediated sensory behaviors
further by testing for genetic interactions of osm-10(n1602) with mutations in other osmosensory defective genes (J. Kass and J. Kaplan, unpublished observations). Two new genes were found that
genetically interact with osm-10 (Table 5). Double
heterozygotes [e.g., osm-10(n1602)/+ IIIC; eos-1(nu288)/+
IV or osm-10(n1602)/eos-2(nu268) III] are defective
for osmotic avoidance. All of these mutations are recessive as single
heterozygotes [e.g., osm-10(n1602)/+, eos-1(nu288)/+, or eos-2(nu268)/+], and all are
normal for osmotic avoidance. This type of genetic interaction, termed
nonallelic noncomplementation, is often seen with genes involved in a
common biochemical pathway. Consistent with this hypothesis,
eos-2(nu268)/+; eos-1(nu288)/+ animals are
defective in osmotic avoidance. Yet, eos-1 and
eos-2 mutant animals are morphologically normal and indistinguishable from wild type for other behaviors examined, including chemotaxis, volatile repellent avoidance, nose touch response, dauer formation, male mating, locomotion, and egg laying. Expression and subcellular localization of OSM-10 is unchanged in
eos-1(nu288) animals. Expression of OSM-10 in
eos-2(nu268) and interactions of eos mutations
with nr2076 are under examination (A. C. Hart,
unpublished observations) These results suggest that osm-10,
eos-1, and eos-2 are part of a common signal
transduction pathway for ASH-mediated osmosensation.
| |
DISCUSSION |
We have shown that ASH-mediated osmotic and touch sensitivities
are mediated by distinct signaling pathways. First, habituating the
nose touch response has no effect on osmotic or 1-octanol avoidance.
This result demonstrates that animals distinguish nose touch from other
ASH sensory modalities. Second, mutations in osm-10 disrupt
ASH-mediated osmosensation but not ASH-mediated touch or volatile
sensitivities. The specificity of the sensory defects observed in
osm-10(nr2076) clearly demonstrates that these pathways are distinct.
This result is somewhat surprising because the responses to
hyperosmolarity and touch are both thought to be mediated by
mechanically gated ion channels. Examples include mechanically gated
channels in the cochlea, putative cytoskeletal-anchored ion channels in C. elegans, and ubiquitous stretch-activated channels in
osmosensation (Oliet and Bourque, 1993, 1996). Hence, a single receptor
could have functioned in both sensory modalities analogous to the dual function proposed for the capsaicin receptor in chemical and thermal sensation (Caterina et al., 1997). On the other hand, separate receptors would facilitate differentiation of disparate stimuli. Although touch is a ubiquitous and relatively innocuous stimulus, osmotic shock and volatile chemicals are lethal. Thus, animals may need
to distinguish between these stimuli. Stimulus strength may, in part,
underlie ASH modality encoding.
Relationship between ASH and microtubule
mechanoreceptor neurons
A molecular model for the touch cell mechanoreceptor has been
proposed by Chalfie and colleagues (Garcia-Anoveros et al., 1995; Gu et
al., 1996). We found that none of the mec genes that constitute this putative mechanoreceptor are required for the ASH-mediated touch of osmotic responses. In addition, the degenerins deg-1 and unc-8, proposed to be mechanoreceptors
on the basis of their homology to mec genes (Chalfie et al.,
1993), were not required for ASH-mediated responses. These results
suggest that the mechanoreceptors used by ASH are distinct from those
used by the touch cells, although mec or degenerin genes
could play redundant roles in ASH. In contrast, the analysis of
mammalian osmosensing neurons suggests that amiloride-sensitive
stretch-activated channels may be required for detecting changes in
osmolarity (Oliet and Bourque, 1993, 1996). It is not surprising that
ASH and the microtubule touch cells might use distinct
mechanoreceptors, because the sensory endings of these cells are
dissimilar ultrastructurally. The ASH sensory endings have ciliary
axonemes (Albert et al., 1981; Perkins et al., 1986), whereas the
microtubule touch cells have an unusual cytoskeleton containing 15 protofilament microtubules (Chalfie and Thomson, 1979). Our results
provide further evidence of the proposal that the mechanoreceptors of
ciliated and microtubule-containing mechanosensory neurons are
distinct, as suggested previously by others (Kernan and Zuker,
1995).
Role of OSM-10 in osmosensation
The selectivity of the sensory defects in osm-10
animals suggests that OSM-10 plays a specific role in transducing
osmosensory signals and is not required for general aspects of ASH
function or development. The specificity of the
osm-10(nr2076) defect suggests that the ASH response to high
osmolarity differs from responses to nose touch and volatile repellents
and that the latter two do not require OSM-10 activity.
Although all living cells respond to changes in osmolarity, the
biochemical mechanisms of osmoregulation are only now being elucidated.
Generally, the response to osmotic shock is twofold, with a relatively
immediate activation of stretch-modulated ion channels and a slower
induction of protein kinase cascades. At present, only one
stretch-activated channel has been molecularly defined, the E. coli channel MscL, which may mediate efflux of organic solutes in
response to osmotic shock (for review, see Sukharev et al., 1997). In
mammalian cells, osmotic shock activates unidentified potassium and
chloride channels (Hallows and Knauf, 1994; Strange et al., 1996). In
both yeast and mammalian cells, hypertonic shock induces the HOG-1 p38
MAP kinase (Brewster et al., 1993; Galcheva-Gargova et al., 1994). In
yeast, the osmosensing MAP kinase cascades are coupled to either of two
alternative osmosensory receptors (Sln1 or Sho1) (Maeda et al., 1994,
1995). In mammalian cells, swelling-activated channel activity is not
blocked by agents that prevent activation of osmosensory MAP kinases,
suggesting that these aspects of the osmosensory response are
mechanistically distinct (Tilly et al., 1996). No MscL, Sln1, or Sho1
homologs have been found in any organism including C. elegans, although the C. elegans genome sequence is
nearly completed. C. elegans MAP kinases and potassium and
chloride channels have not been implicated in osmosensation.
On the basis of these precedents, OSM-10 could play various roles in
osmosensation. OSM-10 might act as a cytoplasmic regulator of the
osmotically activated channels, analogous to pICln,
which regulates swelling-activated chloride channels (Krapivinsky et al., 1994). OSM-10 might act as a chaperonin specifically required for
the trafficking or localization of osmosensory receptor complexes. Alternatively, OSM-10 might act as a cytoplasmic target of an osmosensory receptor, such as Ypd1, which is phosphorylated by the
two-component osmosensor Sln1p in yeast (Maeda et al., 1994; Posas et
al., 1996).
Detecting and responding to changes in external osmolarity are critical
physiological functions of all living cells. Changes in osmolarity
alter cell size and shape-evoking compensatory changes in membrane
permeability to ions and organic solutes. Despite the fundamental
importance of these osmoregulatory effects, relatively little is known
about the molecular mechanisms underlying osmotic regulation of
cellular volume or sensory detection of changes in osmotic regulation
or detection. Further characterization of the osm-10 and
eos genes should provide new insights into the mechanisms
underlying osmosensation.
| |
FOOTNOTES |
Received Dec. 4, 1998; accepted Dec. 22, 1998.
This work was supported by National Institutes of Health Grant NS32196
to J.M.K. and by grants from the Medical and Whitehall foundations to
A.C.H. J.M.K. is a Pew Scholar in the Biomedical Sciences. A.C.H.
is a Searle Scholar. We thank J. Thomas and R. Horvitz for
isolation and initial characterization of osm-10(n1602) and of the role of ASH in osmosensation, the Nemapharm division of Axys
Pharmaceuticals for osm-10(nr2076), C. Bargmann for
plasmids, C. Korey and R. Moeller for assistance with phenotypic
characterization, the C. elegans Genetics Center and M. Chalfie for strains, the C. elegans genome-sequencing
project for clones, and members of the J. M. Kaplan, G. Ruvkun, and S. van den Heuvel laboratories for comments and advice.
Correspondence should be addressed to Dr. Anne C. Hart, Department of
Pathology, Harvard Medical School, and Massachusetts General Hospital
Cancer Center, 149-7202 13th Street, Charlestown, MA 02129.
| |
REFERENCES |
-
Albert PS,
Brown SJ,
Riddle DL
(1981)
Sensory control of dauer larva formation in C. elegans.
J Comp Neurol
198:435-451[Web of Science][Medline].
-
Avery L,
Horvitz H
(1989)
Effects of killing identified pharyngeal neurons on feeding behavior in Caenorhabditis elegans.
Neuron
3:473-485[Web of Science][Medline].
-
Bargmann CI,
Thomas JH,
Horvitz HR
(1990)
Chemosensory cell function in the behavior and development of Caenorhabditis elegans.
Cold Spring Harb Symp Quant Biol
55:529-538[Abstract/Free Full Text].
-
Brewster J,
de Valoir T,
Dwyer N,
Winter E,
Gustin M
(1993)
An osmosensing signal transduction pathway in yeast.
Science
259:1760-1763[Abstract/Free Full Text].
-
Caterina M,
Schumacher M,
Tominaga M,
Rosen T,
Levine J,
Julius D
(1997)
The capsaicin receptor: a heat-activated ion channel in the pain pathway.
Nature
389:816[Web of Science][Medline].
-
Chalfie M,
Thomson JN
(1979)
Organization of neuronal microtubules in the nematode C. elegans.
J Cell Biol
82:278-289[Abstract/Free Full Text].
-
Chalfie M,
Driscoll M,
Huang M
(1993)
Degenerin similarities.
Nature
361:504[Medline].
-
Colbert H,
Smith T,
Bargmann C
(1997)
OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans.
J Neurosci
17:8259-8269[Abstract/Free Full Text].
-
Culotti JG,
Russell RL
(1978)
Osmotic avoidance defective mutants of the nematode Caenorhabditis elegans.
Genetics
90:243-256[Abstract/Free Full Text].
-
Driscoll M,
Chalfie M
(1991)
The mec-4 gene is a member of a family of C. elegans genes that can mutate to induce neuronal degeneration.
Nature
349:588-593[Medline].
-
Driscoll M,
Kaplan J
(1997)
Mechanotransduction.
In: C. elegans II (Riddle D,
Blumenthal T,
Meyer B,
Priess J,
eds), pp 645-677. Cold Spring Harbor, NY: Cold Spring Harbor.
-
Galcheva-Gargova Z,
Derijard B,
Wu I,
Davis R
(1994)
An osmosensing signal transduction pathway in mammalian cells.
Science
265:806-808[Abstract/Free Full Text].
-
Garcia-Anoveros J,
Ma C,
Chalfie M
(1995)
Regulation of Caenorhabditis elegans degenerin proteins by a putative extracellular domain.
Curr Biol
5:441-448[Medline].
-
Gu G,
Caldwell GA,
Chalfie M
(1996)
Genetic interactions affecting touch sensitivity in Caenorhabditis elegans.
Proc Natl Acad Sci USA
93:6577-6582[Abstract/Free Full Text].
-
Hallows K,
Knauf P
(1994)
Principles of cell volume regulation.
In: Cellular and molecular physiology of cell volume regulation (Strange K,
ed), pp 3-29. Boca Raton, FL: CRC.
-
Hart A,
Kramer H,
Van Vactor D,
Paidhunghat M,
Zipursky SL
(1990)
Induction of cell fate in the Drosophila retina: the bride of sevenless protein is predicted to contain a large extracellular domain and seven transmembrane segments.
Genes Dev
4:1835-1847[Abstract/Free Full Text].
-
Hart A,
Sims S,
Kaplan J
(1995)
A synaptic code for sensory modalities revealed by analysis of the C. elegans GLR-1 glutamate receptor.
Nature
378:82-85[Medline].
-
Huang M,
Chalfie M
(1994)
Gene interactions affecting mechanosensory transduction in C. elegans.
Nature
367:467-470[Medline].
-
Kaplan JM,
Horvitz HR
(1993)
A dual mechanosensory and chemosensory neuron in C. elegans.
Proc Natl Acad Sci USA
90:2227-2231[Abstract/Free Full Text].
-
Kernan M,
Zuker C
(1995)
Genetic approaches to mechanosensory transduction.
Curr Opin Neurobiol
5:443-448[Web of Science][Medline].
-
Krapivinsky G,
Ackerman M,
Gordon E,
Krapivinsky L,
Clapham D
(1994)
Molecular characterization of a swelling-induced chloride conductance regulatory protein, pICln.
Cell
76:439-448[Web of Science][Medline].
-
Maeda T,
Wurgler-Murphy S,
Saito H
(1994)
A two-component system that regulates an osmosensing MAP kinase cascade in yeast.
Nature
369:242-245[Medline].
-
Maeda T,
Takekawa M,
Saito H
(1995)
Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor.
Science
269:554-558[Abstract/Free Full Text].
-
Maricq AV,
Peckol E,
Driscoll M,
Bargmann C
(1995)
glr-1, a C. elegans glutamate receptor that mediates mechanosensory signaling.
Nature
378:78-81[Medline].
-
Nonet M,
Staunton J,
Kilgard M,
Fergestad T,
Hartwieg E,
Horvitz H,
Jorgensen E,
Meyer B
(1997)
Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles.
J Neurosci
17:8061-8073[Abstract/Free Full Text].
-
Oliet S,
Bourque C
(1993)
Mechanosensitive channels transduce osmosensitivity in supraoptic neurons.
Nature
364:341-343[Medline].
-
Oliet S,
Bourque C
(1996)
Gadolinium uncouples mechanical detection and osmoreceptor potential in supraoptic neurons.
Neuron
16:175-181[Web of Science][Medline].
-
Perkins LA,
Hedgecock EM,
Thomson JN,
Culotti JG
(1986)
Mutant sensory cilia in the nematode Caenorhabditis elegans.
Dev Biol
117:456-487[Web of Science][Medline].
-
Posas F,
Wurgler-Murphy S,
Maeda T,
Witten E,
Thai T,
Saito H
(1996)
Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor.
Cell
86:865-875[Web of Science][Medline].
-
Strange K,
Emma F,
Jackson P
(1996)
Cellular and molecular physiology of volume-sensitive anion channels.
Am J Physiol
270:C711-C730[Abstract/Free Full Text].
-
Sukharev S,
Blount P,
Martinac B,
Kung C
(1997)
Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities.
Annu Rev Physiol
59:633-657[Web of Science][Medline].
-
Sulston JE,
Schierenberg E,
White JG,
Thomson JN
(1983)
The embryonic cell lineage of the nematode Caenorhabditis elegans.
Dev Biol
100:64-119[Web of Science][Medline].
-
Tavernarakis N,
Shreffler W,
Wang S,
Driscoll M
(1997)
unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion.
Neuron
18:107-119[Web of Science][Medline].
-
Tilly B,
Gaestel M,
Engel K,
Edixhoven M,
de Jonge H
(1996)
Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade.
FEBS Lett
385:133-136.
-
Troemel E,
Chou H,
Dwyer N,
Colbert H,
Bargmann C
(1995)
Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans.
Cell
83:207-218[Web of Science][Medline].
-
White JG,
Southgate E,
Thomson JN,
Brenner S
(1986)
The structure of the nervous system of Caenorhabditis elegans.
Philos Trans R Soc Lond [Biol]
314:1-340.
Copyright © 1999 Society for Neuroscience 0270-6474/99/1961952-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
R. K. Hukema, S. Rademakers, and G. Jansen
Gustatory plasticity in C. elegans involves integration of negative cues and NaCl taste mediated by serotonin, dopamine, and glutamate
Learn. Mem.,
October 30, 2008;
15(11):
829 - 836.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. A. Hallem and P. W. Sternberg
Acute carbon dioxide avoidance in Caenorhabditis elegans
PNAS,
June 10, 2008;
105(23):
8038 - 8043.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. T. Wragg, V. Hapiak, S. B. Miller, G. P. Harris, J. Gray, P. R. Komuniecki, and R. W. Komuniecki
Tyramine and Octopamine Independently Inhibit Serotonin-Stimulated Aversive Behaviors in Caenorhabditis elegans through Two Novel Amine Receptors
J. Neurosci.,
December 5, 2007;
27(49):
13402 - 13412.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Juo, T. Harbaugh, G. Garriga, and J. M. Kaplan
CDK-5 Regulates the Abundance of GLR-1 Glutamate Receptors in the Ventral Cord of Caenorhabditis elegans
Mol. Biol. Cell,
October 1, 2007;
18(10):
3883 - 3893.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. V. Gabel, H. Gabel, D. Pavlichin, A. Kao, D. A. Clark, and A. D. T. Samuel
Neural Circuits Mediate Electrosensory Behavior in Caenorhabditis elegans
J. Neurosci.,
July 11, 2007;
27(28):
7586 - 7596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. A. Bates, M. Victor, A. K. Jones, Y. Shi, and A. C. Hart
Differential contributions of Caenorhabditis elegans histone deacetylases to huntingtin polyglutamine toxicity.
J. Neurosci.,
March 8, 2006;
26(10):
2830 - 2838.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Faumont and S. R. Lockery
The Awake Behaving Worm: Simultaneous Imaging of Neuronal Activity and Behavior in Intact Animals at Millimeter Scale
J Neurophysiol,
March 1, 2006;
95(3):
1976 - 1981.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Y. Chao, H. Komatsu, H. S. Fukuto, H. M. Dionne, and A. C. Hart
Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit
PNAS,
October 26, 2004;
101(43):
15512 - 15517.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Syntichaki and N. Tavernarakis
Genetic Models of Mechanotransduction: The Nematode Caenorhabditis elegans
Physiol Rev,
October 1, 2004;
84(4):
1097 - 1153.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Solomon, S. Bandhakavi, S. Jabbar, R. Shah, G. J. Beitel, and R. I. Morimoto
Caenorhabditis elegans OSR-1 Regulates Behavioral and Physiological Responses to Hyperosmotic Environments
Genetics,
May 1, 2004;
167(1):
161 - 170.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zhang, I. Sokolchik, G. Blanco, and J. Y. Sze
Caenorhabditis elegans TRPV ion channel regulates 5HT biosynthesis in chemosensory neurons
Development,
April 1, 2004;
131(7):
1629 - 1638.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Frank, P. D. Baum, and G. Garriga
HLH-14 is a C. elegans Achaete-Scute protein that promotes neurogenesis through asymmetric cell division
Development,
December 29, 2003;
130(26):
6507 - 6518.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Liedtke, D. M. Tobin, C. I. Bargmann, and J. M. Friedman
Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans
PNAS,
November 25, 2003;
100(suppl_2):
14531 - 14536.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
L. Bianchi, S.-M. Kwok, M. Driscoll, and F. Sesti
A Potassium Channel-MiRP Complex Controls Neurosensory Function in Caenorhabditis elegans
J. Biol. Chem.,
March 28, 2003;
278(14):
12415 - 12424.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. W. Faber, C. Voisine, D. C. King, E. A. Bates, and A. C. Hart
Glutamine/proline-rich PQE-1 proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine neurotoxicity
PNAS,
December 24, 2002;
99(26):
17131 - 17136.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Nolan, T. R. Sarafi-Reinach, J. G. Horne, A. M. Saffer, and P. Sengupta
The DAF-7 TGF-beta signaling pathway regulates chemosensory receptor gene expression in C. elegans
Genes & Dev.,
December 1, 2002;
16(23):
3061 - 3073.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Kass, T. C. Jacob, P. Kim, and J. M. Kaplan
The EGL-3 Proprotein Convertase Regulates Mechanosensory Responses of Caenorhabditis elegans
J. Neurosci.,
December 1, 2001;
21(23):
9265 - 9272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. X. Liu, J. M. Spoerke, E. L. Mulligan, J. Chen, B. Reardon, B. Westlund, L. Sun, K. Abel, B. Armstrong, G. Hardiman, et al.
High-Throughput Isolation of Caenorhabditis elegans Deletion Mutants
Genome Res.,
September 1, 1999;
9(9):
859 - 867.
[Abstract]
[Full Text]
|
|
|
|
Top
Abstract
Introduction
