a glial k/cl transporter controls neuronal receptive...
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Article
A Glial K/Cl Transporter Controls Neuronal
Receptive Ending Shape by Chloride Inhibition ofan rGCGraphical Abstract
Highlights
d Glia discriminate among their associated neurons
d Glia impact neuron shape/function by control of the neuronal
ionic microenvironment
d Chloride ions are inhibitory ligands of receptor guanylyl
cyclases
d cGMP inhibits sensory microvilli shape through WASP and
independent of CNG channels
Singhvi et al., 2016, Cell 165, 936–948May 5, 2016 ª2016 Elsevier Inc.http://dx.doi.org/10.1016/j.cell.2016.03.026
Authors
Aakanksha Singhvi, Bingqian Liu,
Christine J. Friedman, Jennifer Fong,
Yun Lu, Xin-Yun Huang, Shai Shaham
In Brief
Glia regulate the shape and function of
restricted subsets of the neurons they
ensheath by modulating the ionic
microenvironment, thereby controlling
specific aspects of animal behavior.
Article
A Glial K/Cl Transporter ControlsNeuronal Receptive Ending Shapeby Chloride Inhibition of an rGCAakanksha Singhvi,1 Bingqian Liu,2 Christine J. Friedman,1,3 Jennifer Fong,1,4 Yun Lu,1 Xin-Yun Huang,2
and Shai Shaham1,*1Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA2Department of Physiology, Weill Medical College, Cornell University, 1300 York Avenue, New York, NY 10065, USA3Present address: St. George’s University School of Medicine, Grenada, West Indies4Present address: SUNY Downstate Medical Center, College of Medicine, 450 Clarkson Avenue, Brooklyn, NY 11203, USA
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.cell.2016.03.026
SUMMARY
Neurons receive input from the outside world orfrom other neurons through neuronal receptive end-ings (NREs). Glia envelop NREs to create special-ized microenvironments; however, glial functionsat these sites are poorly understood. Here, wereport a molecular mechanism by which glia controlNRE shape and associated animal behavior. TheC. elegans AMsh glial cell ensheathes the NREsof 12 neurons, including the thermosensory neuronAFD. KCC-3, a K/Cl transporter, localizes specif-ically to a glial microdomain surrounding AFDreceptive ending microvilli, where it regulates K+
and Cl� levels. We find that Cl� ions function asdirect inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclicguanosine monophosphate (cGMP). High cGMPmediates the effects of glial KCC-3 on AFD shapeby antagonizing the actin regulator WSP-1/NWASP.Components of this pathway are broadly expressedthroughout the nervous system, suggesting thationic regulation of the NRE microenvironment maybe a conserved mechanism by which glia controlneuron shape and function.
INTRODUCTION
Neurons receive information from the environment or other
neurons through dendritic structures termed neuronal receptive
endings (NREs). In the mammalian CNS, postsynaptic neurons
receive excitatory inputs at NREs termed spines, actin-rich
receptive endings that protrude from the dendrite shaft. Devel-
opmental and experience-dependent remodeling suggests that
plasticity of spine morphology may correlate with learning and
memory (Bourne and Harris, 2008). Perturbations in spine shape
are associated with disorders, including epilepsy, dementia,
schizophrenia, Huntington’s disease, Alzheimer’s disease, and
936 Cell 165, 936–948, May 5, 2016 ª2016 Elsevier Inc.
fragile X syndrome (Penzes et al., 2011). Spines are often en-
sheathed by astrocytic glia (Chung et al., 2015). Glial cues are
implicated in spine shape control; however, mechanisms by
which they regulate spine morphology in vivo are not well under-
stood (Christopherson et al., 2005; Chung et al., 2015; Murai
et al., 2003).
Sensory NREs are comprised of microtubule-based cilia or
actin-based microvilli and are also glia approximated. Sensory
NRE shape perturbation leads to sensory deficits and is common
in patients with congenital defects, such as deafness, blindness,
Usher’s syndrome, or inherited conditions, such as retinal degen-
eration. Genetic lesions underlying these syndromes affect
sensory organ glia or associated neurons/neuron-like cells (Es-
trada-Cuzcano et al., 2012; Kremer et al., 2006). Glial mediators
of sensory NRE shape are not known.
Glia often provide trophic support for neurons, complicating
the investigation of their roles in NRE shape control in vivo.
Caenorhabditis elegans AMsh glia, which envelop neurons of
the amphid sense organ, resemble mammalian glia but are
dispensable for neuron survival. Thus, they are an excellent
model to study glial control of NRE shape in vivo (Shaham,
2010). The AMsh glial cell ensheathes microvilli NREs of the
AFD thermosensory neuron, as well as NREs of 11 other
neurons, including the ciliated NRE of the AWC chemo-
sensory neuron (Figure 1A) (Ward et al., 1975). We have
previously shown that ablation of AMsh glia disrupts AFD
microvilli and AWC cilia NREs (Bacaj et al., 2008). Whether
this reflects passive or active glial roles was unclear. Further-
more, molecules mediating glial contribution to NRE shape
were unknown.
AFD NREs consist of microvilli and a single simple cilium
(Figure 1A) (Doroquez et al., 2014; Perkins et al., 1986). Biogen-
esis of C. elegans cilia has been explored (Inglis et al., 2007).
However, mutations in genes affecting cilium development
only weakly perturb AFD-dependent thermosensation and do
not affect the length, number, or distribution of AFD microvilli
(Figure S1A) (Perkins et al., 1986; Tan et al., 2007). Conversely,
mutations in the gene ttx-1 block microvilli formation and
disrupt temperature sensation, but leave the AFD cilium intact
(Procko et al., 2011; Satterlee et al., 2001). Thus, different
molecular programs control cilium and microvilli structures,
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Tcultivation: 15oC
Tcultivation: 25oC
Figure 1. Glial KCC-3 Controls AFD Neuron Receptive Ending Shape
(A) AMsh glia ensheathes multiple neurons. AFD (orange) also has a cilium (blue). The boxed region magnified in Figure 7.
(B) Histogram depicting AFD defects in indicated genotypes. Numbers, animals scored. Error bars, SEM. ***p < 0.0002. For transgenics, three independent lines
were scored. Green text, glia expression; red text, AFD expression. Second bar, non-transgenic siblings.
(C and D) Fluorescence images of wild-type (C) and kcc-3(ok228) (D) AFD microvilli. Scale bar, 1 mm. Arrow, dendritic shaft. Arrowhead, microvilli.
(E) kcc-3 gene structure; the deletions used are indicated by red and blue bars. Orange, sequences not included in WormBase annotation.
(F) Histogram details are as in (B). Time, hr post-mid-L4 stage.
(G and H) Thermotaxis behavior assays for indicated genotype raised at 15�C (G) and 25�C (H). Animals in (G), 24 hr post-mid-L4 stage. Animals in (H), 96 hr post-
mid-L4 stage. N, number of animals.
See also Figures S1 and S2.
and microvilli are important for AFD-mediated thermosensa-
tion. Shape control of microvilli NREs is poorly understood in
any system.
Here, we report a mechanism by which a sense organ glial
cell continuously regulates the shape of a microvilli-based
sensory NRE and its associated animal behavior by regulating
the NRE microenvironment. We find that an AMsh glia-
expressed K/Cl co-transporter, KCC-3, regulates AFD NRE
shape and C. elegans thermosensory behavior by controlling
Cl� levels surrounding AFD NREs. Cl� ions directly inhibit
the AFD neuron-specific receptor guanylyl cyclase (rGC)
GCY-8 by binding to the conserved S(x)nGPxC motif in its
extracellular domain. GCY-8 determines cyclic guanosine
monophosphate (cGMP) levels within AFD, along with the
phosphodiesterases PDE-1 and PDE-5. High cGMP levels
antagonize the actin regulator WSP-1/WASP, blocking NRE
growth.
While KCC-3 affects AFD neuron shape, it is not required for
AWC neuron NREmaintenance. KCC-3 localizes to a glial micro-
domain surrounding the AFDNRE, but not around the AWCNRE.
Thus, a single glial cell discriminates between the different neu-
rons with which it associates.
Homologous and analogous components of this pathway are
expressed throughout the CNS/peripheral nervous system of
many species. Our results suggest that ionic modulation of
receptor activity at NREs may be a conserved mechanism by
which glia regulate NRE shape and function.
RESULTS
Glia-Secreted and/or Membrane-Bound Factors ControlAFD NRE ShapeTo determine whether AMsh glia provide merely passive struc-
tural support or whether active signaling promotes AFD NRE
shape acquisition, we aimed to determine whether glia-secreted
or membrane-bound proteins are required for AFD microvilli
shape control. We generated animals containing a dominant-
negative version of the endoplasmic reticulum-Golgi trafficking
regulator RAB-1 (RAB-1S25N) under control of the AMsh glia-spe-
cific and temperature-sensitive ver-1 promoter. This construct is
predicted to block exocytic traffic of secreted and membrane
proteins at 25�C (Satoh et al., 1997). Thus, transient incubation
at 25�C for 24 hr leaves glia structurally intact but should impair
signaling. Using this tool, we blocked glial secretion in young
Cell 165, 936–948, May 5, 2016 937
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***
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24h 96h
KCl NaCl K-acetate KCl NaCl K-acetate KCl-acute
11556
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(p678)cng-3(jh113)
tax-4;cng-3 kcc-3(ok228); tax-4
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*****
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kcc-3::GFPPAMsh glia::mCherry
KCC-3::mCherryPAFD::GFP
* *
n.s.
n.s.
tlasontlason
Figure 2. KCC-3 Localizes to Glial Membranes Surrounding AFD and Regulates KCl Homeostasis
(A and B) Fluorescence images depicting expression of indicated transcriptional reporters. Box, cells of interest. Circles + line in (B) indicate distinct cells.
(C) Histogram details as in Figures 1B and 1F. +, presence of kcc-3(ok228) allele. **p = 0.009.
(D and E) Arrow, AFD receptive ending region. Asterisk, location of AWC cilium. Scale bar, 5 mm. Dotted line in (D) outlines AMsh glia and in (E) outlines the animal.
(F and G) Histogram details are as in Figures 1B and 1F. **p < 0.02. ns, not significant compared to age-matched kcc-3(ok228). Hatched bars, animals with
disorganized AFD microvilli.
adults. A pronounced defect in AFD and AWC NRE shape was
observed, mimicking the effects of glia ablations (Figures 1B
and S2A). By contrast, non-transgenic animals raised at 15�Cand transferred as L4 larvae to 25�C or transgenic animals raised
at 15�C (Figure 1B) have normal AFDmicrovilli. Thus, secreted or
membrane-bound glial cues are required continuously to main-
tain AFDmicrovilli shape post-development. Glia-dependent dy-
namic plasticity of sensory structures had not been previously
appreciated.
KCC-3 Is a Regulator of AFD NRE Shape and FunctionTo identify the relevant glial cues controlling AFD microvilli
shape, we performed candidate mutant and RNA interference
screens focused on inactivating glia-enriched genes encoding
membrane or secreted proteins (Bacaj et al., 2008). We found
that mutations in the kcc-3 gene result in AFD microvilli loss
(Figures 1C–1F). KCC-3 encodes a predicted K/Cl co-trans-
porter homologous to human solute carrier protein SLC12A4,
a protein predicted to require RAB-1-mediated trafficking
for membrane localization. A null lesion in the gene, kcc-
3(ok228), results in temperature- and age-dependent loss
of AFD NREs. Ninety-six hour kcc-3(ok228) adults or kcc-
3(ok228) adults grown at 15�C display more pronounced defi-
cits than 24 hr adults or adults grown at 25�C, respectively (Fig-
ure 1F). Weaker but similar defects accompany kcc-3(tm3649)
mutants, which harbor a smaller in-frame truncation of the gene
938 Cell 165, 936–948, May 5, 2016
(Figures 1E and 1F), consistent with the severity of gene
disruption.
AFD is the primary thermosensory neuron inC. elegans, and its
NREs house proteins mediate thermosensory transduction (Garr-
ity et al., 2010). This transduction apparatus is also required for the
ability of C. elegans to remember its temperature of cultivation
(Mori and Ohshima, 1995). We found that kcc-3(ok228) animals
exhibit deficits in thermotaxisbehaviorwhenasked tochoose their
cultivation temperatureona temperaturegradient.Thesebehavior
defects mirror the temperature and age-dependence of kcc-
3(ok228) AFD NRE defects (Figures 1G, 1H, and S2B), suggesting
a common underlyingmechanismbywhich KCC-3 regulates AFD
receptive ending shape and AFD-mediated animal behavior.
The behavior and NRE shape defects we observed in kcc-
3(ok228) mutants are similar to those reported for AMsh glia-
ablated animals (Bacaj et al., 2008), suggesting that KCC-3 is a
major mediator of the glial effect on AFD.
KCC-3 Functions in AMsh Glia to Regulate AFD NREShapeTo confirm that KCC-3 is expressed in AMsh glia, we examined
animals carrying a kcc-3 (50UTR::gfp::30UTR) transgene (Tanis
et al., 2009). kcc-3 expression is detected exclusively in glia,
including AMsh glia, but not in AFD (Figures 2A and 2B). Expres-
sion of kcc-3 coding sequences under the same 50 UTR, or
expression of a kcc-3 cDNA under control of a heterologous
AMsh glia-specific promoter, rescued kcc-3(ok228) AFD neuron
shape defects (Figure 2C). Thus, KCC-3 functions specifically in
AMsh glia to control AFD NRE shape.
KCC-3 Localization Is Restricted to a Glial MicrodomainSurrounding AFD NREsAMsh glia ensheath sensory NREs of other neurons, such
as AWC, in addition to those of AFD. Furthermore, AMsh glia
ablation or exocytosis block with RAB-1S25N disrupts the NRE
structure of these neurons (Figure S2A) (Bacaj et al., 2008).
Thus, a general mechanism may underlie NRE shape control
by glia. Surprisingly, however, we found that kcc-3 mutations
do not affect AWC NREs (Figures S2A, S2C, and S2D). To inves-
tigate the origin of this specificity, we examined KCC-3 subcellu-
lar localization using a genomic fosmid clone containing the
kcc-3 locus recombineered with GFP immediately upstream of
the kcc-3 stop codon or a clone in which a kcc-3 cDNA is tagged
with mCherry and expressed under an AMsh glia-specific pro-
moter. We found that KCC-3 localizes to the portion of the
AMsh glia apical domain in which AFD microvilli are embedded
and is conspicuously absent from the region surrounding AWC
or any other amphid neuron (asterisk, Figures 2D and 2E).
Restricted localization, therefore, likely explains the specific
effects of KCC-3 on AFD receptive endings.
Thus, contrary to our initial assumption, the AMsh glial cell
employs different molecular mechanisms to control AFD and
AWC receptive ending shape.While single glial cells in other sys-
tems also contact multiple neurons, whether they discriminate
between these neurons has been a major outstanding question.
Our results provide definitive in vivo evidence that they can do so.
Glial KCC-3 Regulates AFD NRE Shape by Regulating K+
and Cl� LevelsKCC channels transport K+ and Cl� ions across membranes
(Russell, 2000). We wondered, therefore, whether ionic imbal-
ance may underlie AFD defects in kcc-3(ok228) mutant animals.
To test this, we compared AFD receptive ending defects in kcc-
3(ok228) mutants cultivated on standard C. elegans growth me-
dium and those raised on plates supplemented with 150 mM
KCl. Remarkably, AFDmicrovilli morphology in kcc-3(ok228) mu-
tants raised on high KCl was largely normal (Figure 2F). Rescue
of the kcc-3 mutant defects could also be achieved acutely, as
72-hr animals transferred to KCl-supplemented plates showed
significant rescue within 24 hr (Figure 2F; ‘‘KCl-acute’’).
To determine whether both K+ and Cl� were required for
rescue, we raised kcc-3(ok228) mutants on plates supplemented
with 150 mM Na+Cl� or K+acetate�. While modest rescue was
seen in younger animals in both cases, this was not sustained
in older animals (Figure 2F).
Thus, K+ and Cl�, regulated by KCC-3, are acutely required to
control AFD microvilli shape. Furthermore, the specific localiza-
tion of KCC-3 to glial membranes around AFD strongly suggests
that these ions function in the vicinity of the AFD NRE.
KCC-3 Regulates AFD NRE Shape through the AFD-Specific rGC, GCY-8AFD neuronal activity requires a cyclic nucleotide-gated (CNG)
channel composed of the TAX-2 b subunit and the TAX-4 or
CNG-3 a subunits (Cho et al., 2004; Coburn and Bargmann,
1996; Komatsu et al., 1996). CNG channels allow cations (Na+,
K+, and Ca+) to flow acrossmembranes down their electrochem-
ical gradients. Our finding that medium supplementation with
K-acetate fails to rescue kcc-3(ok228) mutants suggested that
impaired K+ conductance through these cation channels is
unlikely to fully explain the AFD NRE defects of these mutants.
To test this directly, we examined tax-4 and cng-3 singlemutants
or tax-4;cng-3 double mutants. We found that these animals
have only minor defects in AFD microvilli length, with some
exhibiting elongated and disorganized microvilli (Figure 2G;
hatched bars). Furthermore, mutations in genes functioning in
downstream interneurons (ttx-3) or in adaptation of other neu-
rons (egl-4) also have no effect on AFD shape (Figure S1B).
Thus, the effects of KCC-3 on AFD shape cannot be entirely
explained by reduced CNG channel conduction and appear
independent of downstream circuit dynamics.
We considered the possibility that glial KCC-3 defects could
lead to increased K+ and Cl� accumulation in the extracellular
spacesurroundingAFDmicrovilli, leading tohyperactivesignaling
through CNG channels. If this were the case, CNG channel muta-
tions should mitigate the AFD shape defects of kcc-3(ok228)
mutants. However, we found that tax-4 mutations did not sup-
press kcc-3(ok228) defects at all (Figure 2G). Our data, therefore,
suggest that neuronal activity is not the primary effector of KCC-3
for AFD microvilli shape control. We therefore sought another
neuronal effector.
CNG channels are activated by cGMP, synthesized by recep-
tor guanylyl cyclases (rGCs). rGCs are type I transmembrane
proteins with extracellular (ECD), transmembrane (TM), kinase-
homology (KHD), hinge (H), and guanylyl cyclase catalytic
(CAT) domains (Figure 3A). The AFD neuron expresses the
rGCs GCY-8, GCY-18, and GCY-23, which function redundantly
to regulate thermotaxis behavior (Inada et al., 2006; Yu et al.,
1997). We examined gcy-8(tm949), gcy-18(nj38), and gcy-
23(ok797) single loss-of-function mutants and found no defects
in AFD receptive ending shape. Remarkably, however, the gcy-
8(tm949) lesion, predicted to eliminate GCY-8 cyclase activity,
strongly mitigates the AFDmicrovilli defects of kcc-3(ok228) mu-
tants (Figures 3B and S3). This effect is largely specific to gcy-8,
as the gcy-23(ok797) loss-of function allele only weakly rescues
the AFD NRE defects of kcc-3(ok228) mutants (Figure 3B).
Intriguingly, a genomic fosmid, in which GFP sequences are
fused just upstream of the gcy-8 stop codon, is expressed spe-
cifically in AFD, and GFP fluorescence is localized to AFDmicro-
villi (Figure S4A), consistent with previous studies.
Together, our studies suggest that GCY-8 is a key effector
of KCC-3, and loss of KCC-3 results in an increase in GCY-8
activity.
Increased GCY-8 Activity, through KHD Inactivation,Blocks AFD NRE ExtensionThe hypothesis that increased GCY-8 activity mediates the
effects of glial kcc-3 lesions was serendipitously corroborated
by results of a genetic screen we conducted seeking mutants
with defective AFDmicrovilli. From 10,800 F2 progeny examined
(3.5 genomes), we recovered six relevant mutants. Genetic map-
ping, whole-genome sequencing, and transformation rescue
Cell 165, 936–948, May 5, 2016 939
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Figure 3. The Neuronal rGC GCY-8 Functions Downstream of glial KCC-3
(A) GCY-8 domain structure.
(B) Histogram are details as in Figures 1B and 1F. *p = 0.34.
(C) Partial alignment of GCY-8. Red, conserved glycine mutated in gcy-8(ns335).
(D, F, and H) Fluorescence (D), TEM (F), and FIB-SEM (H) reconstruction, respectively, of gcy-8(ns335) AFD receptive-ending. (D) Arrow, dendritic shaft.
Arrowhead, short microvilli. (F) Arrowheads, microvilli. Asterisk, dendritic shaft. Arrow, glial cytosol. Scale bar, 1 mm.
(E and G) TEM (E) and FIB-SEM (G) reconstruction, respectively, of wild-type AFD receptive-ending. (E) Arrowheads, microvilli. Asterisk, dendritic shaft. Arrow,
glial cytosol. Scale bar, 1 mm.
(I) Histogram details as in Figure 1B.
(J and K) Thermotaxis assays as in Figure 1G. Animals raised at 15�C (J) or 25�C (K).
(L) Histogram as in Figure 1B. Hatched, animals with disorganized microvilli.
See also Figures S3 and S4 and Movies S1, S2, S3, and S4.
studies revealed that one of these, ns335, has a causal lesion in
gcy-8. The gcy-8(ns335) mutation is predicted to cause a G707E
change in a highly conserved D(F/H/Y)G motif within the kinase
homology domain (Figures 3A, 3C, and S3A). This lesion has
not been previously described in other rGCs.
Fluorescence microscopy revealed that gcy-8(ns335) mutants
have fewer and shorter AFD microvilli (Figure 3D), and we
confirmed this using serial-section transmission electron micro-
scopy (TEM) (Figures 3E and 3F) and focused ion beam scanning
EM(FIB-SEM) (Figures3Gand3H;MoviesS1,S2,S3, andS4).Us-
ing TEM, we found that while wild-type AFD neurons have 43 ± 2
microvilli (n = 6),gcy-8(ns335)mutants have12± 1 (n=4)microvilli.
SEM imageanalysis showedmaximalmicrovilli lengthof2.5mmfor
wild-type animals, but only 1.5 mm for gcy-8(ns335).
gcy-8(ns335)/+ heterozygotes do not show AFD NRE abnor-
malities, and the defects of gcy-8(ns335) homozygotes are
940 Cell 165, 936–948, May 5, 2016
rescued by expression of wild-type genomic sequences (Fig-
ure 3I), demonstrating that ns335 is a recessive allele. However,
gcy-8(tm949) mutants, lacking the GCY-8 cyclase domain, have
no effect on AFD microvilli shape and suppress kcc-3(ok228)
mutant defects (Figure 3B). Thus, gcy-8(ns335) is unlikely to be
a loss-of-function allele.
Supporting this idea, while gcy-8(tm949) mutants have near-
normal thermotaxis behavior on a linear thermal gradient (Was-
serman et al., 2011), gcy-8(ns335) mutants accumulate at low
temperatures (15�C), regardless of their cultivation temperature
(Figures 3J and 3K). Furthermore, unlike gcy-8(tm949), the
gcy-8(ns335) allele does not suppress the AFD NRE shape de-
fects of kcc-3(ok228) (Figure S4B). Thus, gcy-8(ns335) appears
to be a gain of GCY-8 function. The recessive nature of gcy-
8(ns335) could be explained by the observation that rGC
dimerization is essential for function (Potter, 2011). Formation
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-111
ns335:
0
20
40
60
80
100
1 2 3 4 5 6 7 8
226 104198
113
- + - + - +-146 75 103
******
161
+ns335:
B
% a
nim
als
with
sho
rt vi
lli
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9
C
100 127 46 94 151 41 14259
- + -+ - +- -
**
115-
******
ns335:% a
nim
als
with
def
ectiv
e vi
lli
Figure 4. Excess GCY-8 and cGMP Cause
Loss of AFD-Receptive Ending
Histogram details are as in Figure 1B. +/ �, pres-
ence or absence, respectively, of gcy-8(ns335).
Diagrams depict overexpressed GCY-8 protein.
(A) Excess cyclase activity induces AFD microvilli
loss. White-blue, weak-strong predicted cyclase
activity.
(B) KHD domain inhibits cyclase activity.
(C) Excess cGMP from human rGCs induces AFD
microvilli loss. **p < 0.02. n.s., not significant.
of heterodimeric mutant/wild-type GCY-8 complexes in gcy-
8(ns335)/+ heterozygotes would be predicted to reduce cyclase
activity. Indeed, expression of a cyclase-dead GCY-8D976A pro-
tein in AFDcan rescue gcy-8(ns335) microvilli defects (Figure 4A),
presumably by promoting non-productive dimer formation as is
the case for equivalent mutations in other rGCs (Thompson and
Garbers, 1995).
The gcy-8(ns335) mutation results in a G707E substitution in a
conserved D(F/H/Y)G motif in the kinase-homology domain
(KHD) of GCY-8 (Figures 3A, 3C, and S3A). Deletion of the
KHD of the human rGC NPR1 enhances cyclase activity in cell-
culture experiments (Koller and Goeddel, 1992). Thus, the gcy-
8(ns335) mutation may block the inhibitory effect of the KHD
domain on cyclase activity. To understand the role of the KHD,
we first developed an in vivo assay for
GCY-8 function. Specifically, we found
that overexpression of a GCY-8 cDNA
in AFD neurons of otherwise wild-type
animals using an AFD promoter::gcy-8
cDNA plasmid injected at high copy pro-
motes microvilli loss (Figure 4A). Thus,
increased GCY-8 activity leads to AFD
NRE loss. Furthermore, expression of
full-length GCY-8 containing an E1057A
mutation, whose equivalent hyper-acti-
vates other rGCs (Wedel et al., 1997),
weakly but significantly exaggerated
the effects of wild-type GCY-8 overex-
pression (Figure 4A). We also gener-
ated wild-type animals expressing the
cyclase-dead GCY-8D976A protein. This
single amino-acid substitution curtailed
the ability of GCY-8 overexpression to
block microvilli growth (Figure 4A).
To test the effect of the KHD domain on
cyclase activity, we first showed that
overexpressing a fragment of GCY-8 con-
sisting only of the HCAT domain was
sufficient to elicit AFD microvilli defects
(Figure 4A). Similar constructs for other
rGCs have constitutive cyclase activity in
cell-culture studies (Thompson andGarb-
ers, 1995). As expected if gcy-8(ns335)
is a gain-of-function allele, expression of
the hyper-activated GCY-8 HCAT frag-
ment fails to rescue gcy-8(ns335) defects (Figure 4A). However,
AFD microvilli NRE loss was abrogated if the HCAT domain
was fused to the KHD domain, with or without the transmem-
brane domain (KHD-HCAT or TM-KHD-HCAT) (Figure 4B). Of
note, while the TM-KHD-HCAT protein rescues gcy-8(ns335),
the KHD-HCAT protein fragment does so significantly less effi-
ciently (Figure 4B), suggesting that membrane localization of
GCY-8 is important for its dimerization and cyclase activity regu-
lation. Our results suggest that elevated cyclase activity, affected
by inhibition of the KHD domain, blocks AFD NRE extension.
Our findings also suggest that the conserved DFG motif,
mutated in gcy-8(ns335) animals and present in the KHD of all
rGCs, is likely the relevant element mediating cyclase inhibition
by the KHD domain in vivo.
Cell 165, 936–948, May 5, 2016 941
0
20
40
60
80
100
1 2 3
174
% a
nim
als
with
de
fect
ive
mic
rovi
lli
GCY-8 (S110E) (C141S)117 87
***
0
20
40
60
1 2 3 4 5 6
% a
nim
als
with
co
llaps
ed m
icro
villi
KCl NaCl174 169 82
HCATKCl NaCl
146 70 75
0
20
40
60
80
100
1 2 3
-20
0
20
40
60
80
100
120
A
0.0
2.0
4.0
6.0
1 2 3 4 5 6 7 8
23oC
[cG
MP
] fol
d ch
ange
ov
er h
NP
R-1
wild
type
G70
7E
E10
57A
E10
06A
G68
0E
GCY-8 hNPR1
B
wild
type
***
******
***
0
20
40
1 2
C
pMol
cGM
P/m
l/mg
of
mem
bran
e pr
otei
n
D
control GCY-8
K+Cl-NH4
+Cl-
% m
axim
al a
ctiv
ity o
f G
CY-
8 m
embr
anes
Salt Concentration10 10030 3003
control with K+Cl-N=3
GCY-8
EF
37oC
GCY-8 (S110E) (C141S)
% b
asal
act
ivity
co
mpa
red
to G
CY-
8
HG N=3
0
50
100
150
200
0 0.5 1 1.5 2 2.5
% m
axim
al a
ctiv
ity
Salt Concentration (mM)10 10030 3003
GCY-8(E1057A)(G707E)
(S110E)
(C141S)
***
** ***
n.s.
*
Figure 5. Chloride Inhibits GCY-8 Cyclase
Basal Activity
(A) Western blot showing expression of indicated
rGC proteins in HEK239T cells.
(B) Histogram showing steady-state cGMP levels
in HEK239T cells expressing the indicated con-
structs. Error bars, SEM of three to four experi-
ments. ***p < 0.0008.
(C) cGMP measurements in membrane fractions
of control HEK293T cells or GCY-8-expressing
HEK293T cells.
(D) Inhibition of GCY-8 membrane fraction gua-
nylyl cyclase activity by chloride. Dotted lines, half-
maximal effect. x axis is plotted on a log10 scale.
(E and F) Histogram details as in Figure 1B. (F)
Mutations engineered in full-lengthGCY-8 noted in
color. See also Figure S5C.*p < 0.03.
(G) Histogram details as in (C). Mutations noted as
in (F). ***p = 0.004; **p = 0.01.
(H) Details as in (D) and (F). Activity of each protein
normalized to basal activity in 0 mM NH4+Cl�
(dashed gray line).
See also Figure S5.
GCY-8 Functions Downstream of KClAs is the case for kcc-3 mutants, the effect of gcy-8(ns335) on
AFD microvilli structure is largely independent of AFD neuron
function, as tax-4 mutations fail to suppress gcy-8(ns335) AFD
microvilli defects (Figure 3L). Importantly, dietary supplementa-
tion with KCl does not rescue gcy-8(ns335) NRE shape defects
(Figure S4B).
Thus, rescue of kcc-3(ok228) NRE shape defects by exoge-
nous KCl is not due to non-specific effects on neuron
morphology. Furthermore, GCY-8 functions downstream of
KCC-3 and KCl, as would be predicted for a KCC-3 effector.
GCY-8 Has a Ligand-Independent Basal Cyclase ActivityInhibited by the DFG MotifOur finding that glial kcc-3 mutant defects can be overcome by
inactivating GCY-8 in AFD neurons suggests that GCY-8 may
942 Cell 165, 936–948, May 5, 2016
have constitutive cyclase activity that is
inhibited by KCC-3. Consistent with this
idea, we found that overexpression of
either GCY-8 or the orphan human retinal
rGC GUCY2D, previously shown to have
basal activity (Duda et al., 1996; Shyjan
et al., 1992), promotes AFD microvilli
loss in wild-type animals (Figure 4C).
However, expression of the human rGC
NPR-1, which requires ANP peptide
for activity, or the rGC scavenger recep-
tor, NPR-3, lacking a cyclase domain,
had negligible effects on AFD shape
(Figure 4C).
To directly test whether GCY-8 has
basal activity, we generated stably inte-
grated HEK293T cell lines expressing
wild-type or mutant versions of hNPR-1
or GCY-8 proteins and assayed them for
steady-state cGMP levels (Figures 5A and 5B). We draw three
important conclusions from these studies. First, while hNPR1
has essentially no basal activity, as previously reported, GCY-
8-expressing cells show significant activity, as predicted by
our genetic data. Second, consistent with our in vivo studies,
GCY-8G707E, which affects the conserved DFG motif in the
KHD domain, enhances this basal cyclase activity, similar to or
greater than an activating lesion previously described in other
rGCs (GCY-8E1057A). Third, the GCY-8G707E analogous mutation
in hNPR1 (hNPR1G680E) also shows increased cyclase activity.
Thus, GCY-8 has basal cyclase activity, and the glycine residue
of the DFG motif is important for cyclase inhibition across rGCs.
Cl� Ions Inhibit GCY-8Our data raise the possibility that KCC-3 may inhibit GCY-8
through the action of K+ orCl�. To testwhether this effect is direct
and which ion can inhibit GCY-8 activity, we performed reconsti-
tuted membrane fraction assays using the stably integrated
HEK293T cell lines described above. As with the whole-cell as-
says, we found a 2.7-fold increase in cGMP production in GCY-
8-containing fractions (Figure 5C). Strikingly, we found that Cl�
ions, but not K+ or other anions, are potent inhibitors of GCY-8
cyclase activity and cGMP production (Figures 5D and S5A),
with an IC50 of�60mM.This IC50 iswithin the physiological range
for extracellular Cl� concentrations in many settings (Tora et al.,
2015), suggesting that changes in extracellular Cl�, as would
be predicted to occur in kcc-3 mutants, should indeed affect
GCY-8 activity. Consistent with these in vitro results, we found
that exogenous supplementation with Cl� mitigates the overex-
pression defects of GCY-8 in vivo (Figure 5E). Also, previous
studies suggest that cGMP levels may be reduced at lower tem-
peratures to allow CNG channels in AFD to close as part of the
thermosensory response (Garrity et al., 2010). Thus, defects in
inhibition of cGMP production by GCY-8 should be more pro-
nounced at 15�C, which is exactly what we see in null kcc-3mu-
tants (Figure 1F). Taken together, our results support a model in
which KCC-3 regulates extracellular Cl� levels, which, in turn,
directly influence GCY-8 guanylyl cyclase activity.
Cl� Inhibits GCY-8 by Binding to a FunctionallyConserved Cl� Binding Motif in Its ECDCrystal structures of the rat rGC NPR1 reveal a Cl� in the ECD
domain within a pocket defined by the motif S(x)nGPxC (van
den Akker et al., 2000; Ogawa et al., 2010). A conserved S(x)nGPxC motif is present in the ECD of GCY-8 (Figures S5B and
S5C), but not in GCY-23, mutations in which suppress kcc-3 le-
sions only modestly (Figures 3B and S5C). The S(x)nGPxC motif
is structurally conserved in the ECD of metabotropic glutamate
receptors (mGluRs), and Cl� is an orthosteric ligand for mGluRs
(DiRaddo et al., 2015; Tora et al., 2015). Of note, the IC50 for Cl�
inhibition of GCY-8 is identical to the Cl� EC50 measured for
mGluR2 activity, consistent with a possible role for the GCY-8
S(x)nGPxC ECD motif in mediating the effects of Cl�.Supporting this idea, exogenous in vivo supplementation with
Cl� salts does not suppress the overexpression defects of the
HCAT fragment lacking the ECD, but suppresses defect of full-
length GCY-8 overexpression (Figure 5E). Furthermore, mutating
the conserved Cl� binding domain serine of mGluRs to gluta-
mate mimics the effects of Cl� (Dutzler, 2003; Tora et al.,
2015). We found that a similar mutation in GCY-8 (GCY-8S110E)
inhibits the ability of GCY-8 to promote NRE involution in vivo
(Figure 5F), suggesting that this motif may indeed be functionally
conserved. To confirm this, we expressed GCY-8S110E in
HEK293T cells and measured basal guanylyl cyclase activity in
reconstituted membrane fractions. GCY-8S110E had reduced
basal activity compared to GCY-8, as expected of a mutation
that mimics binding of an inhibitory Cl� ligand (Figure 5G).
Furthermore, GCY-8S110E was insensitive to increasing levels
of Cl�, unlike full-length GCY-8 (Figure 5H). By contrast, the
GCY-8G707E- and GCY-8E1057A-activated proteins that possess
intact ECDs retain Cl� inhibition, but with dampened efficacy
compared to full-length GCY-8 (Figure 5H).
Changing the conserved cysteine of the S(x)nGPxC motif to
serine enhances Cl� coordination in mGluRs by introducing a
second hydroxyl moiety into the Cl� binding pocket (Figures
S5B and S5C) (Tora et al., 2015). We found that the same may
be true for the S(x)nGPxCmotif of GCY-8. GCY-8C141S slightly re-
duces the ability of GCY-8 to prevent NRE extension in vivo (Fig-
ure 5F) and shows reduced basal activity compared to GCY-8
(Figure 5G) and reduced Cl� sensitivity in vitro (Figure 5H).
Taken together, these results suggest that Cl� ions directly
inhibit GCY-8 activity by binding to the conserved S(x)nGPxC
motif in the extracellular domain of GCY-8.
Excess cGMP Blocks AFD NRE GrowthOur studies suggest the hypothesis that increased cGMP
production by GCY-8 promotes AFD NRE disappearance. To
examine the involvement of cGMP in AFD NRE shape control,
we reasoned that GCY-8-independent manipulations that alter
cGMP levels within AFD should also alter microvilli morphology.
Therefore, we expressed cyclase-activated forms of human
NPR-1 (hNPR-1E1006A) and human GUCY2D (hGUCY2DE1010A,
a retinal rGC) in the AFD neuron (Wedel et al., 1997). As shown
in Figure 4C, transgenic animals display defects in microvilli
extension, suggesting that excess cGMP produced by these
heterologous guanylyl cyclases indeed blocks AFD NRE growth.
Similarly, the hGUCY2D protein containing a lesion homologous
to the GCY-8G707E lesion (hGUCY2DG686E) also significantly
shortens AFD receptive ending microvilli (Figure 4C).
As an alternate means of modulating cGMP levels, we exam-
ined the consequences of changing the activities of phosphodi-
esterase (PDE) genes, encoding proteins that degrade cGMP, on
AFD receptive ending morphology. C. elegans has four such
genes (pde-1, pde-2, pde-3, and pde-5) (Liu et al., 2010), two
of which (pde-2 and pde-5) are known to be expressed in AFD
(Wang et al., 2013). While a pde-1 gene reporter containing
limited 50 regulatory sequences fused to GFP was not previously
detected in AFD (Wang et al., 2013), we found that a fosmid
derived from genomic DNA surrounding the locus and recombi-
neered to introduce GFP coding sequences just upstream of the
pde-1 stop codon is expressed in the cell (Figure S6C). Thus, at
least three PDEs are expressed in AFD.
A quadruple mutant inactivating all cGMP PDE genes showed
complete loss of AFD microvilli (Figure 6A). A pde-5 pde-1 dou-
ble mutant also displayed a fully penetrant loss of AFD receptive
endings (Figure 6A). Single, double, or triple combinations of mu-
tations in the other cGMP PDE genes or in the cAMP-specific
PDE gene, pde-4, had no effect on AFD receptive ending shape
(Figure S6A). Thus, PDE-1 and PDE-5 are the only PDEs required
for AFD microvilli extension and appear to function redundantly.
We conclude that PDE-1 and PDE-5 likely act cell autonomously
in the AFD neuron to regulate receptive ending shape and that
excess cGMP blocks microvilli extension.
To test whether increasing PDE activity reciprocally promotes
AFDmicrovilli elongation, we examined pde-5 pde-1 double mu-
tants expressing a cDNAderived from the pde-1BmRNA isoform
specifically in AFD (Figure S6B). This transgene strongly rescues
the AFD defects of the double mutant (Figure 6A), supporting
the idea that PDE-1 and PDE-5 exercise interchangeable cell-
autonomous activities required for AFDmorphogenesis. Further-
more, PDE-1B overexpression restores AFD NRE microvilli
to kcc-3(ok228) and gcy-8(ns335) mutants, consistent with the
Cell 165, 936–948, May 5, 2016 943
0
20
40
60
80
100
1 2 3 4 5 6
0
20
40
60
80
100
1 2 3 4 5
A
0
20
40
60
80
100
1 2 3 4 5 6 7 8PDE-1B:
68211811041 161
***
105120
- +- - + - +
***
***
B
% a
nim
als
with
def
ectiv
e vi
lli
wsp-1(gm324)
gcy-8 (ns335)
27188165
***
122161
C
% a
nim
als
with
def
ectiv
e vi
lli
0
20
40
60
80
100
1
98
% a
nim
als
with
def
ectiv
e vi
lli
gcy-8(tm949);gcy-18(ok797)
98
+
WSP-1A WSP-1A WSP-1fosmid
bin16oC 26oC
E
D
% o
f ani
mal
s%
of a
nim
als
wild typewsp-1(gm324)
1153
1328
408
759
Tcultivation: 15oC
Tcultivation: 25oC
0
20
40
60
80
100
1 2 3 4 5 6F
% o
f ani
mal
s0
20
40
60
80
100
1 2 3 4 5 6
323
253
Tcultivation: 25oC
Figure 6. The Actin Regulator WSP-1 Regulates AFD Shape Downstream of GCY-8
(A–C) Histogram details as in Figure 1B. (A) +/�, presence or absence, respectively, of PDE-1B overexpression. (B) Gray, animals with extended microvilli. (C)
Dotted, animals with slightly shorter, but not absent, microvilli.
(D–F) Thermotaxis assays as in Figure 1G. 24 hr post-mid-L4 stage animals raised at 15�C (D) or 25�C (E), and 96 hr post-mid-L4 stage animals raised at 25�C (F).
See also Figures S6 and S7.
idea that high cGMP levels block microvilli elongation in these
mutants (Figure 6A). Importantly, we noticed that in some ani-
mals overexpressing PDE-1B, microvilli are longer than in wild-
type and are sometimes misshapen (Figures 6A, gray bars; Fig-
ures S6D and S6E). Our results suggest, therefore, that cGMP
levels are sufficient to dictate the extent of microvilli elongation.
cGMP Antagonizes the Actin Regulator WSP-1 toControl AFD Receptive Ending ShapeSince the effects of cGMP on AFD NRE shape are largely
independent of CNG channels, we sought to identify a relevant
mediator for shape determination. From a screen of candidate
effectors, we found that overexpression in otherwise wild-type
AFD neurons of a cDNA corresponding to the gene wsp-1,
encoding the well-studied actin regulator NWASP, results in
elongated AFDmicrovilli (Figures 6B, S7A, and S7B), suggesting
that WSP-1 promotes microvilli formation. Strikingly, WSP-1
overexpression restores microvilli to gcy-8(ns335) mutants (Fig-
ure 6B). A similar result is obtained using a genomic clone car-
rying the wsp-1 gene (Figure 6B). These observations suggest
that high cGMP inhibits microvilli growth by antagonizing
WSP-1. Supporting the idea that WSP-1 is normally required
for AFD NRE growth, wsp-1(gm324) mutants homozygous for
944 Cell 165, 936–948, May 5, 2016
a loss-of-function mutation perturbing the wsp-1A mRNA iso-
form are defective in AFD microvilli elongation. Moreover, wsp-
1(gm324) animals also show defects in thermotaxis behavior at
all temperatures and ages tested (Figures 6D–6F). Another allele,
wsp-1(gk208630), which only affects the wsp-1B isoform, does
not affect AFD NREs (Figures S7C and S7D).
We note that wild-type animals overexpressingWSP-1A occa-
sionally display shorter microvilli (Figure 6B). One explanation for
thismay be that high levels ofWSP-1 target-free actin to new fila-
ment ends, preventing elongation of existing filaments (Smith
et al., 2013a). Consistent with this hypothesis, double mutants
between gcy-8(tm949) and gcy-23(ok797) null alleles show a
modest reduction in microvilli length, as do some animals over-
expressing PDE-1B (Figures 6A and 6C).
The finding that wsp-1(gm324) animals exhibit a weaker NRE
defect than do gcy-8(ns335) mutants suggests that a second
actin-polymerizing factor also functions downstream of cGMP.
Importantly, however, the ability of WSP-1 overexpression alone
to promote AFD microvilli elongation in wild-type animals and
rescue the defects of gcy-8(ns335) demonstrates that actin
nucleation in AFD actin-rich microvilli is likely the key step being
regulated by the glia-neuron interactions we uncovered. We
therefore conclude that cGMP antagonizes WSP-1 activity,
Figure 7. Model for AFD Neuron Receptive Ending Shape Control by
AMsh Glia
Magnified view of the boxed region in Figure 1A. Glial KCC-3 regulates chloride
in the extracellular milieu around AFD receptive ending. Chloride inhibits
GCY-8 by binding the S(x)nGPxC motif in its ECD. GCY-8, PDE-1, and PDE-5
control cGMP levels, which antagonizes the actin cytoskeleton through
WSP-1 independent of neuronal activity.
which likely regulates nucleation of actin filaments in AFD NREs.
These results also imply that, unexpectedly, we have found that
cGMP regulates at least two independent effectors—CNG chan-
nels and WSP-1, with different functions in the AFD neuron, and
WSP-1 is the key downstream effector for regulation of AFDNRE
shape.
DISCUSSION
Our studies identify a glial regulator of sensory NRE shape and
reveal that glia can dictate NRE shape by controlling the NRE
microenvironment. Our findings support a model (Figure 7) in
which the glial K/Cl transporter KCC-3 localizes around and
controls Cl� levels surrounding AFD microvilli. Cl� inhibits the
AFD-specific rGC GCY-8, which has a conserved Cl� binding
structural motif. GCY-8, along with PDE-1 and PDE-5, modulate
neuron cGMP levels, and cGMP antagonizes WSP-1, which
promotes NRE elongation, presumably through actin nucle-
ation. Importantly, our studies reveal that glia continuously
maintain AFD NRE shape, presumably by regulation of the
microenvironment.
Glia Discriminate among NREsOur findings that KCC-3 localizes around AFD NREs and is
selectively required for their function demonstrate that a single
glial cell discriminates between neurons, with which it associ-
ates by targeting regulators to specific neuron-contact sites.
Mammalian astrocytes can associate with �100,000 NREs
(Chung et al., 2015), and Ca2+ fluxes in astrocytes have localized
features (Khakh and Sofroniew, 2015). We suggest that the
ability to discriminate between associated neurons may be a
universal property of glia. Understanding howKCC-3 localization
is achieved may provide a molecular handle on targeted control
of neurons by glia.
Glia Control Sensory and Synaptic NRE Shape andFunctionHow actin-based microvilli NREs are formed and maintained is
poorly understood. Our results will likely apply to many such
sensory structures. Indeed, glia/glia-like cells regulate the ionic
milieu of diverse actin-based sensory NREs, and human disease
mutations affect these cells (Estrada-Cuzcano et al., 2012; Ha-
mel, 2007). KCC and NKCC co-transporters are expressed in
glia of the ear, retina, and in Schwann cells (Boettger et al.,
2002; Kettenmann and Verkhratsky, 2008), and their disruption
can lead to NRE degeneration (Gallemore et al., 1997; Strauss,
2005). KCC3 loss in humans leads to sensory neuropathy (Kahle
et al., 2015).
rGCs are also widely expressed in sensory and other neuron
types, and retinal GUCY2D and Grueneberg ganglion GC-G
have basal guanylyl cyclase activities similar to GCY-8 (Chao
et al., 2015; Shyjan et al., 1992). Increased GUCY2D activity
leads to defects in photoreceptor outer segment shape in
Leber’s congenital amaurosis with some patient mutations map-
ping to the ECD (Perrault et al., 2000). These effects are reminis-
cent of the effects of GCY-8G707E on AFD NREs.
Our work may also present a paradigm for understanding glial
effects on spine morphology in the CNS. Astrocytic KCC chan-
nels regulate K/Cl levels in the CNS (Kettenmann and Verkhrat-
sky, 2008). Disruption of K/Cl levels contributes to neuronal
dysfunction in Huntington’s disease and epilepsy models (Kahle
et al., 2015; Tong et al., 2014) and to defects in spine shape
(Murmu et al., 2015).
AMsh glia form a bounded compartment around AFD NREs,
allowing, as in synapses, tight control of the NRE milieu. A role
for extracellular Cl� in modulating spine-localized metabotropic
glutamate receptors has been explored (Tora et al., 2015). Our
studies suggest that in addition to providing a diffusion barrier,
glia may actively control synaptic Cl� levels to modulate spine
activity and shape. Supporting this proposition, mGluRs (class
III GPCRs) and rGCs adopt a Leu/IsoLeu/Val binding periplasmic
protein-like (LIVBP) fold in the extracellular domain (Acher et al.,
2011). The S(x)nGPxC Cl� binding motif is conserved across
nervous system LIVBP fold receptors (Acher et al., 2011).
Glial Control of ThermosensationElectrophysiological studies of AFD neurons suggest that high/
low cGMP opens/closes CNG channels upon warming/cooling
(Garrity et al., 2010; Ramot et al., 2008), relative to the cultivation
temperature. Thus, within the operating range of the thermosen-
sory apparatus, a basal level of cGMP must exist that can be
modulated up or down. That GCY-8 has basal activity supports
this idea, as does our observation that kcc-3 mutants exhibit
more pronounced thermosensory deficits at low temperature,
where higher GCY-8 inhibition may be required.
We have previously shown that expression of the VER-1 re-
ceptor tyrosine kinase in AMsh glia is temperature dependent
and independent of the AFD neuron (Procko et al., 2011). It is
possible that modulation of AMsh glia-dependent properties,
Cell 165, 936–948, May 5, 2016 945
such as KCC-3 activity, by temperature may also form part of the
thermosensory apparatus.
Control of rGC ActivityrGCs are prevalent receptors and are important therapeutic tar-
gets (Potter, 2011). Our data highlight unexpected aspects of
rGC structure and function. That neuronal rGCs (e.g., GCY-8
and GUCY2D) have basal activities, but non-neuronal rGCs
(e.g., NPR-1 and NPR-2) do not, suggests the possibility of a
fundamental dichotomy. The identification of Cl� as a GCY-8
inhibitor raises the possibility that rGCs with basal activity, for
which activating ligands have not been identified, may instead
be regulated by inhibitory extracellular cues. Our results suggest
that the relationship between the ECD and KHDmay be inverted
in rGCs with basal activity versus ligand-activated non-neuronal
rGCs.
That Cl� ions influence GCY-8 activity also suggests that other
ions may control rGCs. A recent study exploring rGCs in K+
and I� sensation in C. elegans revealed that stimulus specificity
tracks with receptors ECDs (Smith et al., 2013b). Thus, rGCs
may sense different ions through their ECD.
Finally, we show that a conserved glycine residue in the KHD
has inhibitory roles in GCY-8 and also in human NPR1. The
D(F/H/Y)G motif containing this amino acid is conserved in all
kinases and pseudo-kinases and forms part of the activation
loop. Thus, this domain may facilitate ATP binding, previously
suggested to affect rGC activity (Goraczniak et al., 1992).
EXPERIMENTAL PROCEDURES
C. elegans Methods
Standard culturing and germ-line transformationmethodswere used (Brenner,
1974; Mello and Fire, 1995). Mutants, transgenes, genetic methods, RNAi
methods, and thermotaxis assays are described in the Supplemental Experi-
mental Procedures.
Plasmids
Plasmid construction details are provided in the Supplemental Experimental
Procedures.
Microscopy and Image Processing
Images were collected on an Axioplan 2 microscope (Zeiss) with 633/1.4 na
objective (Zeiss) and dual-band filter set (Chroma, set 51019). Some images
were collected on a DeltaVision Core imaging system (Applied Precision)
with a PlanApo 603/1.42 na or UPLSApo 1003/1.40 na oil-immersion objective
and a Photometrics CoolSnap HQ camera (Roper Scientific). Images were
deconvolved using ImageJ. Electron microscopy methods are described in
the Supplemental Experimental Procedures.
cGMP ELISAs
ELISAs were performed using the Direct cGMP Kit (Enzo Life Sciences, ADI-
900-014). Cells (53 105) were grown on poly-D-lysine-coated plates (Corning
BioCoat, 354414) for 12 hr, rinsed in D-PBS, and incubated in serum-free me-
dium, supplemented with 0.5 mM IBMX, at 23�C or 37�Cwith 5%CO2 for 1 hr.
Cells were lysed in 0.1 MHCl + 0.1% Triton X-100 and assayed for cGMP con-
centration as per the manufacturer’s protocol (Enzo Life Sciences) (Guo et al.,
2007, 2009). Optical density measurements were performed on a BioTek Syn-
ergy NEO using the Gen5 data analysis software. All GCY-8 experiments were
performed in biological quadruplicate and NPR-1 experiments in biological
triplicate. Importantly, the assay measures steady-state accumulation of
cGMP (production minus degradation) and is always an underestimate of
cGMP production.
946 Cell 165, 936–948, May 5, 2016
Membrane assays were carried out as follows: cells were cultured in growth
medium to �95% confluency and washed in 50 mM NH4Ac and 200 mM
sucrose (pH 7) (plus 1 mM IBMX and protease inhibitors). Cells were passed
through a 26-gauge needle several times and sonicated for 10 s. After centri-
fugation at 2,000 rpm for 5min, themembrane fraction was prepared by centri-
fugation at 100,000 3 g for 1.5 hr at 4�C. The membrane pellet was resus-
pended in 50 mM NH4Ac, 200 mM sucrose (pH 7), 1 mM DTT, 10 mM
MgAc2, and 1 mM MnSO4. Membrane preparations were treated with KCl,
KAc, or NH4Cl. GTP (5 mM) was added to start the cyclase reaction. After
30 min at 30�C, membrane preparations were lysed in 0.2 M HCl and assayed
for cGMP.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and four movies and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.03.026.
AUTHOR CONTRIBUTIONS
A.S. and S.S. designed the experiments and wrote the manuscript. A.S. per-
formed all experiments and analyses, except for electron microscopy, which
was performed by Y.L., and membrane fraction cGMP assays, which was per-
formed by B.L. and X.-Y.H. C.J.F. and J.F. assisted A.S. in construct cloning.
ACKNOWLEDGMENTS
We thank Cori Bargmann,MenachemKatz, Michael Koelle, and Piali Sengupta
for reagents, Tarakhovsky lab members for help with cell culture, and Cori
Bargmann and the S.S. lab for comments. William J. Rice at the Simons Elec-
tron Microscopy Center (NYSBC) helped with FIB/SEM imaging. A.S. was an
American Cancer Society postdoctoral fellow (PF-13-083-01-DDC), NIH-T32
institutional postdoctoral fellow (5T32CA967334), and Murray Foundation
fellow. This work was supported in part by grants from the NIH (NS081490
and HD078703) to S.S.
Received: July 15, 2015
Revised: December 4, 2015
Accepted: March 15, 2016
Published: April 7, 2016
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Supplemental Figures
0
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>20025 38 126
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dyf-11 arl-13 bbs-2 osm-6% a
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Figure S1. Cilia or Downstream Circuit Genes Do Not Affect AFD Microvilli Shape, Related to Figure 1
(A and B) Histogram details are as in Figure 1B.
Cell 165, 936–948, May 5, 2016 ª2016 Elsevier Inc. S1
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PAWC::mCherry
kcc-3(ok228); PAWC::GFP
B
D
A
bin16oC 26oC
% o
f ani
mal
s
wild typekcc-3(ok228)
390
1621
Tcultivation: 25oC
Figure S2. Glial Exocytosis Regulates AWC Independent of KCC-3, Related to Figure 1
(A) Histogram details as in Figure 1B.
(B) Thermotaxis behavior assays on 24 hr post mid-L4 stage animals raised at 25�C summarized as detailed in Figure 1G.
(C and D) Fluorescence images of AWC cilium in wild-type (C) and kcc-3(ok228) mutant animals (D).
S2 Cell 165, 936–948, May 5, 2016
ECD
A
tm949ns335 (GGA>GAA)
GCY-8tm949
KHD CATGCY-8
ECD KHD
TM
TM
B
nj38
C
ok797
GCY-18nj38
GCY-23ok797
GCY-18
GCY-23
ECD
ECD
ECD
ECD ?
KHD
KHD
CAT
CAT
TM
TM
Figure S3. rGC Gene Mutations and Predicted Protein Structures, Related to Figure 3
(A–C) In each box, top panel denotes the gene structure, and lower panels denote the putative domain structure for wild-type or predicted mutant proteins as
noted. Gray bars are exons in top panel of each section, white bars are UTR sequences.
Cell 165, 936–948, May 5, 2016 S3
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Pgcy-8::GCY-8::GFP
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villiB
27
KCl
38
NaCl
32
K-acetategcy-8(ns335)
kcc-3(ok228)gcy-8(ns335) gcy-8(ns335)
60
Figure S4. GCY-8 Is Expressed at AFD Microvilli and Functions Downstream of KCl, Related to Figure 3
(A) GCY-8 localization to AFD microvilli receptive endings. Arrow, AFD microvilli (zoomed in inset). Arrowhead, dendrite.
(B) Histogram details as in Figure 1B.
S4 Cell 165, 936–948, May 5, 2016
C
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180
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0 0.5 1 1.5 2 2.5
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ctiv
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CY-
8 m
embr
anes
in
NH
4+ac
etat
e-
10 10030 3003Salt Concentration (mM)
BA
Rat NPR1 : SSENAAGVCSDT--AAPLAAVDLKWEHSPAVFLGPGCRat NPR2 : SSE-LDGACSEY--LAPLRAVDLKLYHDPDLLLGPGCRat NPR3 : DSD-----CGNRALFSLVDRVAAARGAKPDLILGPVCMouse NPR1 : SSENAAGVCSDT--AAPLAAVDLKWEHSPAVFLGPGCHuman NPR1 : SSENALGVCSDT--AAPLAAVDLKWEHNPAVFLGPGCWorm GCY-8 : ISQTG---CGESYE-GVAVAADMYHLQKVKAFIGPYCWorm GCY-23: LNQMG---CGESYE-GVAVGADMYHVQGVRAFIGPYC
Ser53 (S110)
Pro84(P139)
Cys86(C141)
Ion
Figure S5. Cl�, but Not K+, Inhibits GCY-8 Activity, Related to Figure 5(A) As in Figure 5D except that the y axis denotes % activity over GCY-8, instead of inhibition. x axis is plotted on a log10 scale.
(B) Halide ion, shown with a mesh shell, within the conserved Cl- binding structural motif. Ligand explorer rendering of PDB structure 3A3K of rat NPR1 (Ogawa
et al., 2010). Hydrogen bonds are dashed pink lines. Corresponding amino acids in GCY-8 are listed in parentheses. Hydrogen bond from a C141S mutation is
modeled in dashed yellow line.
(C) Alignment of rGCs highlighting the conserved structural motif (colored amino acids) for Cl- binding. Amino acids underlined are labeled in (B).
Cell 165, 936–948, May 5, 2016 S5
0
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pde-5
pde-1 pde-1 pde-1pde-2
pde-3pde-5
pde-4
A
BAB
nj57 0.5kbPDE-1
+++
+++
+++
+
++
++
++
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pde-2pde-5
pde-2
pde-3
pde-2
+
EDC
PAFD::PDE-1B::GFP
P AFD
::mC
herr
y;pd
e-1:
:GFP
PAFD::PDE-1B::GFP
Figure S6. Some pde Alleles Do Not Affect AFD Shape, Related to Figure 6
(A) Histogram details as in Figure 1B.
(B) Detail as in Figure S3.
(C) Fluorescence image depicting expression of PDE-1 in AFD (dotted line).
(D and E) Fluorescence images of wild-type and elongated microvilli, respectively, in animals overexpressing PDE-1B. Yellow dots, wild-type AFD extent. Red
dots, elongated portion. Scale bar, 5 mm.
S6 Cell 165, 936–948, May 5, 2016
BA
Psrtx-1::WSP-1A::SL2::GFP
gm324
A
B
gk206830 (Q210>Stop)0.5kb
WSP-1
D
0
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100
1
47
wsp-1(gk206830)
% a
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villiC
Figure S7. WSP-1 Overexpression Causes Elongated AFD Microvilli, Related to Figure 6
(A and B) Fluorescence micrographs of long AFD microvilli in WSP-1A transgenic animals. Scale bar, 5 mm. Yellow dotted line, typical length of wild-type AFD
microvilli. Red, additional length. White arrow, dendrite. Arrowheads, longer microvilli.
(C) Histogram as in Figure 1B.
(D) Details as in Figure S3.
Cell 165, 936–948, May 5, 2016 S7
Cell, Volume 165
Supplemental Information
A Glial K/Cl Transporter Controls
Neuronal Receptive Ending Shape
by Chloride Inhibition of an rGC
Aakanksha Singhvi, Bingqian Liu, Christine J. Friedman, Jennifer Fong, Yun Lu, Xin-YunHuang, and Shai Shaham
Supplemental Materials for:
A Glial K/Cl Transporter Controls Neuronal Receptive-Ending Shape by Chloride
Inhibition of an rGC
Aakanksha Singhvi1, Bingqian Liu2, Christine J. Friedman1,3, Jennifer Fong1,4, Yun Lu1, Xin-
Yun Huang2, and Shai Shaham1,*
1Laboratory of Developmental Genetics, The Rockefeller University, 1230 York Avenue,
New York, NY 10065 USA
2Department of Physiology, Cornell University Weill Medical College, 1300 York Avenue,
New York, NY 10065 USA
3Current address: St. George’s University School of Medicine, Grenada, West Indies
4Current address: SUNY Downstate Medical Center College of Medicine, 450 Clarkson
Ave, Brooklyn, NY 11203
*To whom correspondence should be addressed: Tel (212) 327-7126, Fax (212) 327-
7129, email [email protected]
Document contains:
-Supplemental Methods
-Supplemental References
Singhvi et al.
2
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
C. elegans methods
C. elegans were cultured as previously described (Brenner, 1974; Stiernagle, 2006).
Bristol N2 strain was used as wild type. For all experiments, animals were raised at 20oC
(or alternate temperatures where noted) for at least two generations without
starvation. Animals were picked as L4 larvae onto fresh plates and assayed 24 hours
later, unless otherwise noted. Integration of extra-chromosomal arrays was performed
using UV with or out without trioxalen (Sigma, T6137). Germ-line transformations by
micro-injection to generate unstable extra-chromosomal array transgenes were carried
out using standard protocols (Mello and Fire, 1995). For dietary supplementation with
salts, sterile 3M solutions of KCl, NaCl or K-acetate were added to a final concentration
of 150mM on plates. Plates were equilibrated for 48 hours before use.
Mutagenesis and mapping of genetic lesions
nsIs228 (Psrtx-1:GFP) animals were mutagenized with 75 mM ethylmethanesulfonate
(EMS, Sigma M0880) for 4 hours at 20oC. 10,800 F2 progeny were screened for AFD NRE
morphology defects on an Axioplan 2 fluorescence microscope (Zeiss) with a 63x/1.4 NA
objective (Zeiss) and dual-band filter set (Chroma, Set 51019). gcy-8(ns335) mapped to a
0.44 cM interval by Hawaiian SNIP-SNP mapping (Wicks et al., 2001). This interval was
refined to 0.13 cM by deficiency mapping and the gene was identified by fosmid rescue
and candidate gene analyses.
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Strains and plasmids
Some strains listed below in Sections A and B were sourced from (a) the CGC, funded by
NIH Office of Research Infrastructure Programs (P40 OD010440), (b) the International C.
elegans Gene Knockout Consortium (C. elegans Gene Knockout Facility at the Oklahoma
Medical Research Foundation, funded by the National Institutes of Health; and the C.
elegans Reverse Genetics Core Facility at the University of British Columbia, funded by
the Canadian Institute for Health Research, Genome Canada, Genome BC, the Michael
Smith Foundation, and the National Institutes of Health) and (c) National BioResource
Project (NBRP), Japan. Recombineered fosmids were obtained from The C. elegans
TransgeneOme Project.
A. Mutants
LG1: pde-1 (nj57), pde-5 (nj49), arl-13(gk513)
LG2: kcc-3(ok228), kcc-3(tm3649), pde-3 (nj59), pde-4(ok1290)
LG3: tax-4(p768), pde-2(tm3098)
LG4: gcy-8(tm949), gcy-8(ns335), gcy-18(nj38), gcy-23(ok797), cng-3(jh113), wsp-
1(gm324), wsp-1(gk206830), egl-4(n478), egl-4(ad450), bbs-2(gk544)
LG5: osm-6(p811), ttx-1(p767),
LGX: dyf-11(mn392), ttx-3(mg158)
B. Integrated transgenes
nsIs228 was the control strain for all non-transgenic animals.
Strain Chromosome Genotype Reference
nsIs228 I Psrtx-1:GFP (Colosimo et al., 2004). Extra-
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chromosomal array from Piali Sengupta’s
lab was integrated in this study.
nsIs143 X pF16F9.3:DsRed (Procko et al., 2011)
nsIs373 X PsrtxB:GFP AFD specific. Generated in this study by
promoter bashing Psrtx-1
kyIs136 X Pstr-2:GFP (Troemel et al., 1999)
oyIs44 V odr-1:RFP (L’Etoile and Bargmann, 2000)
nsIs109 and
nsIs113
pF16F9.3:DT-
A(G53E)
(Bacaj et al., 2008)
C. Extra-chromosomal transgenes and plasmids generated in this study
All transgenic arrays were generated with Pelt-2:mCherry (Armenti et al., 2014) as co-
injection marker with three exceptions. pde-1 and kcc-3 fosmids had Pmig-24:Venus
((Abraham et al., 2007) as co-injection marker; nsIs228 and nsIs373 had Punc-122:RFP
(Miyabayashi et al., 1999) as marker and Pver-1:rab-1 constructs had pRF4 (Gu et al.,
1998) as co-injection marker. All transgenes are injected at 50 ng/ul unless otherwise
noted.
Extra-chromosomal array
(nsEx) number Plasmid Genotype
3936, 3937, 3938, 3939 pAS300 Psrtx-1B:GFP
4236, 4260, 4381 pAS406 Psrtx-1B:mCherry
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- pAS178 Psrtx-1:SL2:GFP
3944, 3945, 3946, 3947 Recombineered
fosmid Pgcy-8:gcy-8:GFP (20 ng/ul)
3565, 3566 WRM0629cG03 Pgcy-8:gcy-8 (10 ng/ul)
3976, 4015, 3977 pAS185 Psrtx-1:GCY-8:SL2:GFP
3973, 3966, 3974 pAS306 Psrtx-1:GCY-8(E974A):SL2:GFP
4189, 4190, 4194, 4162, 4311 pAS400 Psrtx-1:GCY-8(D976A):SL2:GFP
5634, 5635, 5636, 5637 pAS257 Psrtx1:GCY-8(S110A):SL2:GFP
5638, 5639, 5640, 5641 pAS260 Psrtx1:GCY-8(S110E):SL2:GFP
4080, 4063, 4064,4076 pAS330 Psrtx-1:GCY-8 (HCAT):SL2:GFP
4164, 4165, 4166 pAS351 Psrtx-1:GCY-8(KHD):SL2:GFP
4163, 4496, 4497, 4506, 4498 pAS353 Psrtx-1:GCY-8(KHD+HCAT):SL2:GFP
4442, 4443, 4444, 4574 pAS430 Psrtx-1B:GCY-8(TM+KHD+HCAT):SL2:GFP
4040, 4041, 4042, 4043 pAS316 Psrtx-1:hNPR1:SL2:GFP
4037, 4038, 4039 pAS314 Psrtx-1:hNPR3:SL2:GFP
4195, 4188, 4191, 4192 pAS404 Psrtx-1:hGUCY2D:SL2:GFP
4605, 4617, 4580, 4606 pAS457 Psrtx-1:hNPR1(E1006A):SL2:GFP
4621, 4622, 4623 pAS467 Psrtx-1:hGUCY2D(E1010A):SL2:GFP
5062, 5063, 5613, 5614 pAS506 Psrtx1:GUCY2D(G686E):SL2:GFP
4607, 4646, 4624 pAS468 Psrtx-1:PDE1B:SL2:GFP
4730, 4804, 4767 Recombineered Ppde-1:PDE-1:GFP
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fosmid
3282, 3283, 3297 WRM0636cF12 Pwsp-1:wsp-1
4842, 4843 4853, 4854 pAS245 Psrtx-1:WSP-1A:SL2:GFP
2680, 2655, 2769, 2770 pAS125 Pver-1:RAB-1(S25N)
4809, 4838, 4839, 4810 pMK179.1 Pkcc-3:KCC-3 + pF53F4.13:mCherry
LX1161 pJT68A Pkcc-3:gfp
4995, 4996, 4997 Recombineered
fosmid Pkcc-3-1:KCC-3:GFP
5615, 5616, 5617, 5618 pAS250 pF53F4.13:KCC-3 cDNA:SL2:mCherry
(1 ng/ul)
5650, 5651, 5652 pAS265 pF53F4.13:KCC-3 cDNA:mCherry
(1 ng/ul)
n.a. pAS214 pCMV:FLAG:GCY-8
n.a. pAS221 pCMV:FLAG:GCY-8(G707E)
n.a. pAS227 pCMV:FLAG:GCY-8(E1057A)
n.a. pAS222 pCMV:FLAG:hNPR-1
n.a. pAS501 pCMV:FLAG:hNPR-1(G680E)
n.a pAS265 pCMV:FLAG:GCY-8(S110E)
n.a pAS270 pCMV:FLAG:GCY-8(C141S)
Plasmids
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GCY-8 PLASMIDS: cDNA for gcy-8 was PCR amplified from a mixed stage cDNA library in
two segments using an internal Hind3 segment and cloned into pAS178 by triple ligation
using Xma1/Nhe1 sites flanking either end. This generated the pAS185 plasmid, which
was used as template for all subsequent domain manipulations. gcy-8:gfp fusion
plasmids were made by triple ligation of appropriately modified pAS185 fragments to
enable in frame translation with gfp. Mutations were introduced by site directed
mutagenesis in the appropriate fragment and triple ligating with the appropriate
fragment and vector.
HUMAN rGC PLASMIDS: Human NPR1 (BC063304.1), NPR-3 (BC131540.1) and GUCY2D
(BC148421.1) were obtained from the mammalian gene collection through GE
Dharmacon/Thermo Fisher. Each cDNA was cloned into pAS178 by simple or triple
ligation and this was used as template for all subsequent cloning. Mutations were
introduced by site directed mutagenesis in the appropriate fragment and triple ligating
with the appropriate fragment and vector.
PDE-1B PLASMID: The pde-1B isoform was PCR amplified from a mixed stage cDNA
library and ligated into pAS178 using Xma-1/Nhe-1 flanking sites.
WSP-1A PLASMID: The wsp-1a isoform was PCR amplified from pGO101 (gift from
Grigorios Oikinomou, SS lab) and ligated into pAS465 or pAS178 using Xma-1/Sal-1
flanking sites.
KCC-3 PLASMIDS: The kcc-3 expression construct including the 5’ and 3’ UTR sequences
fused in frame to GFP were a gift from Jessica Tanis, Michael Koelle lab. Pkcc-3::kcc-3 was
constructed by amplifying the coding sequence of kcc-3 and inserting it in frame with
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8
the UTR sequences of pJT68A. The first seven of the fifteen introns in this gene were
included in this construct. pAS250, 255 and 265 were generated by amplifying the KCC-3
cDNA from a mixed stage C. elegans cDNA library and inserting into pAS465, pSGEM or
pAS512 respectively using BamH1/Sal1 flanking sites. For pAS265, a single nucleotide
deletion of the stop codon was done by site directed mutagenesis to put the cDNA in
frame with mCherry. The sequence of the cDNA we isolate suggests that 87 nucleotides
of the annotated intron 8 on WormBase are in fact coding sequence. This adds 29 amino
acids of a stretch of conserved residues, including a Cys conserved in all KCC, but not
NKCC genes across species (data not shown and Figure 1E). This finding is consistent
with KCC-3 being a member of the SLC12A4/KCC family.
FLAG:rGC PLASMIDS: All constructs to generate N-terminal FLAG tagged protein were
made by amplifying cDNA sequences in two fragments with appropriate flanking
restriction enzyme sites and triple ligating into the pFLAG:CMV vector (Sigma E6783).
Mutant proteins were generated by subsequent site-directed mutagenesis of wild type
rGC cDNA inserts.
Electron microscopy
For transmission EM, animals were prepared and sectioned using standard methods
(Lundquist et al., 2001). Imaging was performed with an FEI Tecnai G2 Spirit BioTwin
transmission electron microscope equipped with a Gatan 4K · 4K digital camera. Focused
ion beam scanning EM (FIB-SEM) was performed at the NY Structural Biology Center. A
modified high-contrast en bloc staining OTO method (Seligman et al., 1966) was applied
Singhvi et al.
9
to the FIB-SEM specimen preparation. Sodium thiocarbohydrazide (TCH) was used to
bind the primary osmium stain. Then the en-block stain was enhanced by a second
round of osmium fixation. Glutaraldehyde (1%) was added to provide effective protein
crosslinking in tissues. The serial sections prior to the region-of-interest were searched
and confirmed by TEM imaging before the critical portion of the specimen had been
reached. The samples for SEM were mounted on a stub of metal with silver adhesive,
coated with 40-60 nm of Palladium using a Denton vacuum sputter coater. The image
collection was performed by a Dual beam FEI Helios 650 Focused Ion Beam Scanning
Electron Microscope (FIB-SEM) with a CCD camera with field of view of 4096 x 3536
pixels. Serial backscattered scanning electron micrographs were collected from the
specimen surface after each 30 nm layer had been milled away. Beam voltage was 2keV
and beam current was 0.4nA. Accurate image registration was achieved by applying a
scale-invariant features algorithm (Lowe, 1999) to the raw image stacks.
Cell culture
HEK293T cells were grown using standard procedures at 37oC and 5%C02 in DMEM-C
[DMEM media (Gibco 11995-065) supplemented with 10% heat inactivated fetal bovine
serum, L-Glutamine, MEM-NEAA and Penicillin/Streptomycin]. Transfections were
performed using Lipofectamine 2000 (Invitrogen, 11668-027) as per manufacturer’s
protocol. pCMV:GFP:Pac (Campeau et al., 2009) was used as a co-transfection reagent at
1:10 dilution for each test (i.e. pAS) plasmid. Puromycin efficacy was determined by
serial dilution of antibiotic concentration and assaying viability of un-transfected
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HEK293T cells at 24 and 48 hours. GFP positive transfected cells were selected for stable
integrations and maintained in DMEM-C media supplemented with 4ug/ml puromycin
(Sigma P9620). Expression of FLAG tagged constructs was verified by lysing cells in RIPA
buffer (Sigma, R0278) and Western blotting equal amounts of total protein extract as
quantified by Bradford assay (Bio Rad 500-0202). Detection was performed using anti-
FLAG antibody (Sigma, F7425) and Clean-Blot IP detection kit (HRP) reagents (Thermo
Scientific, 21232). Protein expression level of each construct was normalized to
expression of hNPR-1 with alpha-tubulin as a loading control. Westerns were done in
triplicate and averaged for normalization. Experiments were done at normal
physiological temperature for each protein.
Thermotaxis behavior
Thermotaxis assays were performed on a 18°-26°C linear temperature gradient
(Hedgecock and Russell, 1975; Mori and Ohshima, 1995). Animals were synchronized
and the staged progeny were tested on the first day of adulthood. Briefly, animals were
washed twice with S-Basal and spotted onto the center of a 10-cm plate warmed to
room temperature and containing 12 mL of NGM agar. The plate was placed onto the
temperature gradient (17-26oC) with the addition of 5 mL glycerol to its bottom to
improve thermal conductivity. At the end of 45 mins, the plate was inverted over
chloroform to kill the animals and allowing easy counting of animals in each bin. The
plates have an imprinted 6x6 square pattern which formed the basis of the 6
temperature bins. Each data point is the average of 2-5 assays with ~150 worms/assay.
Singhvi et al.
11
RNAi
Plasmids expressing double-stranded RNA (dsRNA) were obtained from the Ahringer
library (Kamath et al., 2003). An empty vector was used as the negative control. RNAi
was performed by plating L4 nsIs228; rrf-3(pk1426) animals onto RNAi bacteria and
allowing them to feed (Timmons and Fire, 1998). 24 hours later, adults were moved to a
fresh plate with RNAi bacteria and their progeny were assayed 3-5 days later for defects
in AFD receptive ending shape.
Statistics
Statistical significances (p-values) were computed using the two-sample proportion Z
test with normal approximation to the binomial at StatCrunch (www.statcrunch.com),
except for Figure 5B where the unpaired two sample t-test was used.
Singhvi et al.
12
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