Genetics: Published Articles Ahead of Print, published on September 15, 2006 as 10.1534/genetics.106.063115
SWAN-1, a C. elegans WD repeat protein of the AN11 family, is a negative
regulator of Rac GTPase function
Yieyie Yang1,3, Jiamiao Lu1, Joel Rovnak2, Sandra L. Quackenbush2, and Erik A.
Lundquist1*
1
Department of Molecular Biosciences
University of Kansas
1200 Sunnyside Avenue
5049 Haworth Hall
Lawrence, KS 66045
2
Department of Microbiology, Immunology, and Pathology
College of Veterinary Medicine and Biomedical Sciences
Colorado State University
1619 Campus Delivery
Fort Collins, CO 80523-1619
3
Current Address:
Dana-Farber Cancer Institute
44 Binney Street
Smith 870
Boston, MA 02115
USA
*Corresponding author ([email protected], Phone 785-864-5853; Fax 785-864-5294)
1
1
Abstract
2
Rac GTPases are key regulators of cell shape and cytoskeletal organization.
3
While some regulators of Rac activity are known, such as GTPase activating
4
proteins (GAPs) that repress Rac activity, other Rac regulators remain to be
5
identified. The novel C. elegans WD-repeat protein SWAN-1 was identified in a
6
yeast-two hybrid screen with the LIM domains of the Rac effector UNC-
7
115/abLIM. SWAN-1 was found to also associate physically with Rac GTPases.
8
The swan-1(ok267) loss-of-function mutation suppressed defects caused by the
9
hypomorphic ced-10(n1993) allele and enhanced ectopic lamellipodia and
10
filopodia formation induced by constitutively-active Rac in C. elegans neurons.
11
Furthermore, SWAN-1(+) transgenic expression suppressed the effects of
12
overactive Rac, including ectopic lamellipodia and filopodia formation in C.
13
elegans neurons, ectopic lamellipodia formation in cultured mammalian
14
fibroblasts, and cell polarity and actin cytoskeleton defects in yeast. These
15
studies indicate that SWAN-1 is an inhibitor of Rac GTPase function in cellular
16
morphogenesis and cytoskeletal organization. While broadly conserved across
17
species, SWAN-1 family members show no sequence similarity to previously-
18
known Rac inhibitors.
19
2
19
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Introduction
Cell and growth cone migration and the establishment and maintenance of
21
cell shape are tightly-regulated processes controlled by extracellular cues and
22
intracellular physiology (DICKSON 2002; RAFTOPOULOU and HALL 2004; TESSIER-
23
LAVIGNE and GOODMAN 1996; VICENTE-MANZANARES et al. 2005; YU and
24
BARGMANN 2001). Rac GTPases of the Rho subfamily are key regulators of cell
25
shape and act in part by controlling the structure and dynamics of the actin
26
cytoskeleton (HALL 1998). Classically, Rac GTPases were identified by their
27
similarity to Rho and were found to induce the formation of veil-like lamellipodial
28
plasma membrane extensions in serum-starved fibroblasts (HALL 1998; OTIENO
29
et al. 2005). In C. elegans neurons, Rac activity promotes the formation of both
30
lamellipodia and filopodia, thin finger-like plasma membrane extensions
31
(STRUCKHOFF and LUNDQUIST 2003). Loss-of-function studies in Drosophila and
32
C. elegans demonstrate that Rac GTPases are required for a wide variety of
33
morphogenetic events, including axon pathfinding and cell migration (DICKSON
34
2001; LUNDQUIST 2003). The GTPase regulatory cycle involves active, GTP-
35
bound Racs which hydrolyze GTP to GDP and thus self-inactivate, as the GDP-
36
bound form is inactive. Guanine nucleotide exchange factors (GEFs) mediate
37
the exchange of GDP for GTP and thus favor the GTP-bound active state of
38
Racs. GTPase activating proteins (GAPs) stimulate the GTPase activity of Racs
39
and thus favor the inactive state (VAN AELST and D'SOUZA-SCHOREY 1997).
40
Another class of Rho GTPase negative regulators are the guanine nucleotide
41
dissociation inhibitors (GDIs), which inhibit Rho GTPase association with the
3
42
plasma membrane where they are active (MICHAELSON et al. 2001; VAN AELST
43
and D'SOUZA-SCHOREY 1997).
44
In C. elegans, three Rac GTPases CED-10, MIG-2, and RAC-2 have
45
compensatory, redundant roles in axon pathfinding and in the migration of
46
neurons, embryonic cells during gastrulation, and vulval cells (KISHORE and
47
SUNDARAM 2002; LUNDQUIST et al. 2001; SOTO et al. 2002). The actin-binding
48
protein UNC-115/abLIM, which acts with Racs in axon pathfinding, is required
49
for Rac-induced lamellipodia and filopodia in neurons and UNC-115 itself induces
50
the formation of lamellipodia and filopodia in C. elegans neurons and in serum-
51
starved mammalian fibroblasts (LUNDQUIST et al. 1998; STRUCKHOFF and
52
LUNDQUIST 2003; YANG and LUNDQUIST 2005). UNC-115 might be a downstream
53
cytoskeletal effector of Rac GTPases and might control growth cone lamellipodia
54
and filopodia formation in response to Rac signaling during axon pathfinding.
55
The molecular linkage between Racs and UNC-115 in these events is unclear,
56
and molecules that interact physically with UNC-115 remain to be identified.
57
Furthermore, while some Rac inhibitors are known, it will be important to identify
58
all of the molecules that regulate Rac activity to precisely control distinct
59
morphogenetic events.
60
To identify molecules that act with Rac and UNC-115 in axon pathfinding
61
and cell migration, the LIM domains of UNC-115 were used as bait in a yeast
62
two-hybrid screen of a C. elegans cDNA library. This screen identified a novel,
63
conserved molecule called SWAN-1 (seven WD repeat protein of the AN11
64
family), which consists of at least seven WD repeats and a conserved C-terminal
4
65
tail. In two-hybrid and pull-down assays, SWAN-1 was found to interact with both
66
the UNC-115 LIM domains and with the Racs, suggesting that SWAN-1 is a
67
molecular linker between UNC-115 and the Racs. An array of functional tests in
68
C. elegans, mammalian fibroblasts, and yeast indicate that SWAN-1 represses
69
Rac activity. SWAN-1 displays no similarity to other known Rac negative
70
regulators (e.g. GAPs), indicating that SWAN-1 represents a previously-
71
unidentified class of negative regulators of Rac GTPases.
72
5
72
Results
73
SWAN-1 Interacts Physically with Both the UNC-115 LIM domains and Racs
74
UNC-115 is a cytoskeletal linker molecule composed of a C-terminal actin-
75
binding villin headpiece domain (VHD) and three N-terminal LIM domains (Figure
76
1A) (LUNDQUIST et al. 1998). To identify molecules that bind to the UNC-115 LIM
77
domains, a yeast two-hybrid screen using the three UNC-115 LIM domains as
78
bait was conducted (Figure 1A). From an estimated two million C. elegans
79
cDNAs screened, two cDNAs were isolated that encode a novel protein that here
80
is named SWAN-1 (seven WD-repeat protein of the AN11 family-1). The
81
presence of swan-1 two-hybrid fusions with the UNC-115 LIM domain fusion
82
specifically activated both HIS5 expression (data not shown) and lacZ expression
83
(Figure 1B) in the two-hybrid system (see Materials and methods). Both swan-1
84
cDNAs contained the entire swan-1 open reading frame, suggesting that only the
85
full-length SWAN-1 protein is active in the two-hybrid assay. SWAN-1 interacted
86
specifically with the UNC-115 LIM domains, but not with the remainder of the
87
UNC-115 molecule in the two-hybrid system (data not shown).
88
Because UNC-115 acts genetically downstream of Rac signaling
89
(STRUCKHOFF and LUNDQUIST 2003), it was determined if SWAN-1 could also
90
interact with the three C. elegans Rac molecules in the two-hybrid system.
91
SWAN-1 and each of the three C. elegans Racs, CED-10, MIG-2, and RAC-2,
92
interacted when directly tested in the two-hybrid system (Figure 1B). SWAN-1
93
interacted with both wild-type RAC-2 (Figure 1B) as well as constitutively-active,
94
GTPase-dead RAC-2(G12V) (data not shown). The LIM domains of UNC-115
6
95
did not interact with any of the three Racs in the two-hybrid system, nor did any
96
other region of UNC-115 (data not shown).
97
To confirm the physical interactions detected in the two-hybrid system,
98
coimmunoprecipitation of SWAN-1 with UNC-115 and RAC-2 was assayed in
99
lysates of Hela cells in which tagged versions of the molecules were expressed
100
(Figure 1C and D). EGFP-tagged full-length UNC-115 coimmunoprecipitated
101
with Myc-tagged SWAN-1 (Figure 1C), and MYC::RAC-2 coimmunoprecipitated
102
with FLAG::SWAN-1 (Figure 1D). These coimmunoprecipitation experiments
103
were repeated in HEK293 cells with similar results (data not shown). Thus, the
104
two-hybrid and coimmunoprecipitation data presented here indicate that SWAN-1
105
binds to both the UNC-115 LIM domains and to Rac GTPases.
106
107
SWAN-1 Is a Member of a Novel, Conserved Family of Seven WD-repeat
108
Proteins
109
The complete sequences of the two swan-1 cDNAs isolated in the two-
110
hybrid screen were determined, as were the sequences of the independently
111
derived cDNAs yk343g2 and yk326a5 (provided by Y. Kohara) (Figure 2A and
112
B). The splicing pattern and coding potential for each of the four cDNAs was
113
identical. Reverse transcription PCR was conducted on the 5’ end of the swan-1
114
transcript using a 5’ primer complementary to the trans-spliced leader SL1, which
115
revealed that SL1 was spliced onto the 5’ end transcript after position 7464
116
relative to cosmid F53C11 (Figure 2A and B). cDNA sequences indicated that
7
117
the swan-1 transcript was polyadenylated after position 10306 relative to cosmid
118
F53C11 (Figure 2A and B).
119
The swan-1 cDNAs can encode a 388-residue polypeptide that is
120
predicted to contain at least seven WD-repeat elements (Figure 3A and B) (NEER
121
et al. 1994; SMITH et al. 1999). WD repeats are conserved structural elements
122
that form a "β-propeller" structure, with each blade of the propeller corresponding
123
to one WD repeat (SONDEK et al. 1996). WD repeats have no known enzymatic
124
activity. Rather, they are thought to form a scaffold for protein-protein interaction.
125
Many proteins with seven WD repeats have been identified, including the β-
126
subunit of G proteins (SMITH et al. 1999). However, SWAN-1 is a member of a
127
novel, conserved family of WD-repeat proteins (Figure 3C). The SWAN-1 family
128
is ancient, as obvious SWAN-1 counterparts are found in insects, mammals,
129
protozoa, yeast, and plants (Figure 3C). SWAN-1-family members contain at
130
least seven regions similar to the WD repeat, with a potential eighth region near
131
the C-terminus. SWAN-1-family proteins also contain a conserved C-terminal tail
132
that is not found in Gβ or other WD-repeat proteins (Figure 3C). Generally, the
133
first two predicted WD repeats showed more variability between species than do
134
the others.
135
Two SWAN-1 family members in higher plants have known functions.
136
AN11 from petunia is a cytoplasmic molecule involved in Myc and Myb
137
transcription factor activation and anthocyanin pigment biosynthesis in response
138
to light (DE VETTEN 1997). In Arabidopsis, the SWAN-1-like molecule TTG1 is
139
involved in the Myb-dependent specification of trichome cells on leaves (WALKER
8
140
et al. 1999). However, the Arabidopsis genome also encodes another SWAN-1
141
family member, AtAN11 (Figure 3C), which is more similar to SWAN-1 than is
142
TTG1. The role of AtAN11 is not known, nor are the roles of SWAN-1 family
143
members in other organisms.
144
The swan-1 gene corresponds to the gene F53C11.8 in the C. elegans
145
genome. Immediately downstream of the swan-1 gene is the coding region for a
146
gene that encodes another SWAN-1 family member here named SWAN-2
147
(F53C11.7 in C. elegans genome nomenclature) (Figure 3A). The first exon in a
148
swan-2 cDNA is 114 nucleotides downstream of the site of swan-1
149
polyadenylation (data not shown). SWAN-2 is 45% identical at the amino acid
150
level to SWAN-1 (Figure 3C). RNAi of swan-2 in a swan-1(ok267) had no effect
151
on viability, fertility, or gross morphological and behavioral phenotype.
152
Furthermore, neither RNAi of swan-2 nor a swan-2 deletion allele ok964 had an
153
effect on activated CED-10 and RAC-2 in PDE development (data not shown).
154
155
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swan-1(ok267) suppresses ced-10(n1993)
To determine the role of SWAN-1 in C. elegans, a deletion allele of the
157
gene, called ok267, was obtained from the C. elegans Gene Knockout
158
Consortium (kindly provided by G. Moulder and B. Barstead). The swan-1 region
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from ok267 mutants was amplified by PCR and sequenced to determine the
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breakpoints of the deletion. The 1189-bp ok267 deletion encompassed bases
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7846-9034 relative to cosmid F53C11 (Figure 2A and B). To ensure that the
162
wild-type swan-1 locus had not been duplicated in the ok267 deletion strain, PCR
9
163
was performed on swan-1(ok267) genomic DNA using primers that were
164
predicted to amplify a fragment in wild-type but not in swan-1(ok267) (one primer
165
was complementary to a region removed by ok267). These primers amplified a
166
fragment from wild-type but not from swan-1(ok267) (data not shown).
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The ok267 deletion removed coding region for 223 amino acid residues of
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the 388-residue SWAN-1 polypeptide, including all of WD repeats 2-6 and part of
169
7 (Figure 3A and B). swan-1(ok267) animals were viable and fertile and
170
displayed no apparent phenotype, including gross morphological or behavioral
171
defects. RNA-mediated interference (RNAi) directed against swan-1 had no
172
detectable phenotype. Furthermore, no swan-1 transcript was detected by RT-
173
PCR in swan-1(ok267) mutants (data not shown).
174
Because SWAN-1 interacted physically with UNC-115 and the Racs,
175
double mutants of swan-1(ok267) were built with the rac pathway mutants ced-
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10(n1993, n3246, and n3417) (LUNDQUIST et al. 2001), mig-2(mu28) (ZIPKIN et al.
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1997), unc-73(rh40 and e936) (STEVEN et al. 1998), and unc-115(ky275)
178
(LUNDQUIST et al. 1998). These double mutants were analyzed for gross
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morphological phenotype as well as axon pathfinding of the PDE and amphid
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neurons. In no case were any effects of swan-1(ok267) detected (data not
181
shown).
182
ced-10 Rac mutations cause defects in phagocytosis of cells undergoing
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programmed cell death (REDDIEN and HORVITZ 2000) and defects in the migration
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of the gonadal distal tip cells (LUNDQUIST et al. 2001), resulting in malformed
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gonads. swan-1(ok267) significantly suppressed the cell corpse engulfment
10
186
defects and the gonadal distal tip cell (DTC) migration defects of ced-10(n1993)
187
mutants (Table 1). ced-10(n1993) mutants alone displayed 14.9±3.8 persistent
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cell corpses in the pharyngeal region of late three-fold stage embryos, compared
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to 9.6±4.2 in swan-1(ok267); ced-10(n1993) double mutants (p < 0.001).
190
Furthermore, 24% of ced-10(n1993) mutants displayed misshapen gonads due
191
to DTC migration errors, whereas swan-1(ok267); ced-10(n1993) double mutants
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displayed 7% (p < 0.001). swan-1(ok267) alone had little effect on cell corpse
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engulfment or DTC migration on its own (Table 1). ced-10(n1993) is a
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hypomorphic allele that retains some ced-10 activity (REDDIEN and HORVITZ 2000;
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SHAKIR et al. 2006). That swan-1(ok267) suppressed the defects caused by ced-
196
10(n1993) suggests that the normal role of SWAN-1 might be to repress CED-10
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Rac activity (i.e. in the absence of SWAN-1, the remaining activity of ced-
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10(n1993) is increased). While swan-1 slightly suppressed the hypomorphic
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ced-10(n1993) allele, it did not suppress the maternal-effect lethality or gonad
200
defects of the ced-10(n3417) null allele nor did it suppress the lethality or axon
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pathfinding defects associated with the ced-10(n1993); mig-2(mu28) double
202
mutant (data not shown).
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To confirm that the suppression of ced-10(n1993) was due to the swan-
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1(ok267) deletion, a full-length swan-1::gfp transgene (predicted to encode a full-
205
length SWAN-1::GFP fusion protein; see Materials and Methods) was analyzed
206
for its ability to rescue suppression by swan-1(ok267). RNAi of swan-1 resulted
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in greatly-reduced GFP expression from this transgene, suggesting that the
208
transgene encodes a SWAN-1::GFP molecule (data not shown). The swan-
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1::gfp transgene rescued the effects of swan-1(ok267) suppression in ced-
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10(n1993); swan-1(ok267) mutants (Table 1): transgene-bearing ced-10; swan-1
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animals displayed 15.6±3.4 persistent cell corpses and siblings that did not
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inherit the transgene displayed 11.7±3.6 persistent cell corpses (p < 0.001).
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Furthermore, suppression of DTC migration defects was rescued (29% versus
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12%, respectively; p <0.001) (Table 1).
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Mutations in the mig-2 Rac gene also cause DTC migration defects
216
(LUNDQUIST et al. 2001). swan-1(ok267) did not suppress the DTC migration
217
defects of mig-2(mu28) (Table 1). As opposed to ced-10(n1993), which retains
218
some CED-10 function, mig-2(mu28) is a null allele that likely causes complete
219
loss of mig-2 function. This might explain why swan-1(ok267) did not suppress
220
mig-2(mu28).
221
swan-1(ok267) Enhances Constitutively-Active Rac in Neurons
222
To further test the idea that SWAN-1 is a negative Rac regulator, the
223
effect of swan-1 loss of function on Rac activity in development of the PDE
224
neurons was studied. The PDEs are a pair of ciliated sensory neurons that
225
reside in the posterior-lateral region of C. elegans in the post-deirid ganglion
226
(SHAKIR et al. 2006; WHITE et al. 1986). A single ciliated dendrite extends
227
dorsally from the cell body, and a single axon extends ventrally to the ventral
228
nerve cord, where it branches and runs anteriorly and posteriorly (Figure 4A).
229
Previous studies indicated that constitutively-active Rac molecules,
230
produced by the canonical glycine 12 to valine (G12V) mutation, induce ectopic
231
neurite formation and ectopic lamellipodia and filopodia formation in the PDE
12
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neuron in vivo (Figure 4B) (STRUCKHOFF and LUNDQUIST 2003). swan-1(ok267)
233
enhanced the effects of transgenic ced-10(G12V) and rac-2(G12V) (Figure 4C).
234
For example, rac-2(G12V) caused 65% ectopic axon formation and 8% ectopic
235
lamellipod and filopod formation, whereas swan-1(ok267); rac-2(G12V) animals
236
displayed 100% and 74% of the respective defects. While all effects of swan-
237
1(ok267) and swan-1(RNAi) on CED-10(G12V) and RAC-2(G12V) were
238
significant (p < 0.001), the effects of swan-1 on rac-2(G12V) were more
239
pronounced than those on ced-10(G12V). In addition to increasing the
240
penetrance of rac-2(G12V) and ced-10(G12V) defects, swan-1(ok267) also
241
increased the severity of defects. For example, ced-10(G12V) PDEs generally
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displayed 1-2 ectopic axons, whereas swan-1(ok267)l ced-10(G12V) PDEs often
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displayed 3 or more ectopic axons. Occasionally, so many ectopic axons were
244
present that it became impossible to distinguish which axon was the normal PDE
245
axon. RNAi of swan-1 gave similar but weaker results (Figure 4C), indicating
246
that the effects were due to disruption of swan-1 function. These results
247
demonstrate that swan-1(ok267) enhances the effects of ced-10(G12V) and rac-
248
2(G12V).
249
The mig-2(rh17) mutant harbors an activating G16 mutation (ZIPKIN et al.
250
1997). Neither mig-2(rh17) nor the effects of transgenic expression of MIG-
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2(G16V) were significantly affected by swan-1(ok267) (Figure 4C).
252
SWAN-1(+) Transgenic Expression Represses Constitutively-Active Rac
253
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If SWAN-1 is a negative Rac regulator, overactivity of wild-type SWAN-1
might repress Rac activity. Transgenic expression of wild-type swan-1
13
255
suppressed the effects of ced-10(G12V) and rac-2(G12V) (Figure 4D; see
256
Materials and methods). For example, rac-2(G12V) trangenes averaged 65%
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ectopic PDE axons and 6% ectopic lamellipodia and filopodia, whereas rac-
258
2(G12V) transgenes that also contained wild-type swan-1 DNA averaged 16%
259
and 0% defects, respectively. While all effects of SWAN-1(+) expression on
260
CED-10(G12V) and RAC-2(G12V) were significant (p < 0.001), suppression of
261
ced-10(G12V) was weaker than that of rac-2(G12V). swan-1 coexpression had
262
no detectable effect on mig-2(G16V) (Figure 4D).
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swan-1(ok267) Enhances the Neuronal Effects of Dominant Rac alleles
264
In contrast to ced-10(n1993) and mig-2(mu28), the mutations ced-
265
10(n3246), ced-10(n1993lq20), mig-2(rh17), and mig-2(lq13) all cause axon
266
defects on their own, indicating that they are not simple loss-of-function
267
mutations (SHAKIR et al. 2006). mig-2(rh17) affects the glycine 17 residue of the
268
MIG-2 protein (ZIPKIN et al. 1997) and is likely to result in constitutively-active,
269
GTPase-dead MIG-2 (the equivalent mutation was included in the mig-2(G16V)
270
transgenes). It is unknown whether ced-10(n3246), ced-10(n1993lq20), or mig-
271
2(lq13) are activating or dominant-negative mutations.
272
swan-1(ok267) enhanced the axon defects caused by each of these
273
dominant Rac alleles. ced-10(n3246), ced-10(n1993lq20), mig-2(rh17), and mig-
274
2(lq13) caused a variable PDE axon pathfinding phenotype which was
275
categorized as mild (the axon reaches the ventral nerve cord near the normal
276
position but is misguided) or severe (the axon fails to reach the ventral nerve
277
cord near the normal position) (Figure 5A and B). The percentages of combined
14
278
mild and severe axon defects of swan-1(ok267) double mutants with ced-
279
10(n3246), mig-2(lq13), and mig-2(rh17) were increased, and the severity of all
280
four were increased (p < 0.001; Figure 5C). For example, ced-10(n3246) alone
281
showed 8% axon defects, most of which were of the mild class, and swan-
282
1(ok267); ced-10(n3246) showed 18% defects with 8% of the severe class.
283
swan-1 Is Expressed in Many Cell Types Including Neurons
284
To further understand the role of swan-1 in Rac signaling, the temporal
285
and spatial pattern of swan-1 expression during development was analyzed. Two
286
swan-1 expression transgenes were constructed: a transcriptional reporter
287
consisting of the swan-1 promoter driving green fluorescent protein (gfp)
288
expression; and a translation reporter of the full length swan-1 region fused in
289
frame to gfp such that the a full-length SWAN-1 molecule fused to GFP at the C-
290
terminus is produced (SWAN-1::GFP) (see Materials and methods). Full-length
291
swan-1::gfp expression was first detected at approximately 100 minutes post-
292
fertilization when embryonic transcription begins. Expression was observed in
293
most cells (Figure 6A-C), including neuroblasts and neurons, and persisted
294
throughout development into adulthood. The only cells that did not show swan-
295
1::gfp expression were the intestinal cells and their precursors (Figure 6A-C).
296
SWAN-1::GFP accumulated predominantly in the cytoplasm of all cell types
297
analyzed. The transcriptional reporter transgene displayed a temporal and
298
spatial expression pattern identical to that of the full-length fusion transgene
299
(data not shown).
300
SWAN-1 represses human Rac1 in 3T3 fibroblasts
15
301
The above data indicate that SWAN-1 represses Rac activity in C. elegans
302
neuronal morphogenesis. CED-10 Rac and RAC-2 Rac are very similar to
303
human Rac1 (83% identical at the amino acid level) (LUNDQUIST et al. 2001).
304
Expression of constitutively-active Rac1 (the glutamine to leucine mutation at
305
position 61 (Q61L)) in serum-starved 3T3 fibroblasts led to lamellipodial
306
membrane protrusions along cell edges (Figure 7A). We found that 62% of 3T3
307
cells expressing Rac1(Q61L) exhibited lamellipodial plasma membrane
308
extensions (Figure 7A-B) not seen in egfp control-transfected cells. Expression
309
of myc-tagged SWAN-1 alone had little effect on cell morphology (Figure 7B).
310
Fewer cells transfected with both Rac1(Q61L) and SWAN-1 displayed
311
lamellipodial structures (26% compared to 62% for Rac1 alone; p < 0.001)
312
(Figure 7B), suggesting a partial suppression of Rac1(Q61L) activity by SWAN-1.
313
SWAN-1 might directly repress Rac activity. Alternatively, SWAN-1 might
314
inhibit downstream Rac effectors, such as UNC-115. In C. elegans neurons, no
315
repression of activated UNC-115 by SWAN-1 was detected (data not shown). As
316
previously reported, addition of an N-terminal myristoylation sequence (Myr) from
317
human c-Src targeted UNC-115 to the plasma membrane and caused
318
overactivation of UNC-115 resulting in ectopic lamellipodia and filopodia
319
formation in serum-starved fibroblasts (Figure 7C). In 24% of MYR::UNC-115-
320
expressing cells, lamellipodial and filopodial membrane protrusions were
321
observed (Figure 7C and D). Co-transfection of SWAN-1 with MYR::UNC-115
322
resulted in 15% of cells exhibiting protrusive morphology, a difference that was
323
not statistically significant (p = 0.1076). While these data indicate that SWAN-1
16
324
might slightly repress UNC-115, the effect is not as pronounced as the effect of
325
SWAN-1 on Rac1.
326
SWAN-1 represses Rac activity in yeast
327
In the course of the directed two-hybrid experiments using SWAN-1 and
328
the Racs, it was noted that yeast transformed with CED-10 and MIG-2 (but not
329
RAC-2) were slow-growing and formed small and irregular-shaped colonies. The
330
doubling times of strains harboring CED-10 and MIG-2 grown in liquid culture
331
were increased compared to the parent strain Y190 (Figure 8A). CED-10 cells
332
had a doubling time of greater than 10 hours and MIG-2 cells had a doubling time
333
of about 6 hours, compared to about 2.5 hours for Y190. Doubling time
334
compared to Y190 was not significantly affected by RAC-2 or SWAN-1
335
expression (data not shown). When cell morphology was analyzed, fewer of the
336
CED-10 and MIG-2 cells were in the process of budding during the log phase of
337
growth compared to the parent Y190 strain (Figure 8B). For example, 85% of
338
Y190 cells were in the process of budding whereas 29% of CED-10 and 45% of
339
MIG-2 cells were budding. SWAN-1 and RAC-2 expression had little effect on
340
percentage of cells undergoing budding (Figure 8B).
341
CED-10 and MIG-2-expressing cells displayed abnormal morphology,
342
including greatly-increased cell size and irregular cell shape (18/21 of CED-10-
343
expressing cells and 24/30 MIG-2-expressing cells). Phalloidin staining revealed
344
that the actin cytoskeleton was disorganized in these cells. In normal budding
345
cells, the actin cytoskeleton is dramatically polarized; the newly-forming bud
346
displays actin patches on the cell surface as a result of endocytosis involved in
17
347
bud growth; and long actin filaments extend from the mother cell to the bud
348
(ADAMS and PRINGLE 1984). The actin cytoskeleton of the host strain Y190
349
displayed this morphology characteristic of asymmetric growth and budding
350
(Figure 8C). In CED-10 and MIG-2 cells undergoing budding, actin patches were
351
often observed in both the bud and in the mother cell (Figure 8D and E),
352
indicating that cell polarity and asymmetric growth had been disrupted.
353
Furthermore, the long actin filaments were often missing (Figure 8D and E).
354
When the long filaments were present, they were often disorganized with respect
355
to the bud-mother cell axis (Figure 8D). In the large, irregular, non-budding cells,
356
actin patches were observed uniformly on the cell surface (Figure 8G), indicating
357
that these cells were undergoing non-polarized growth without budding. While
358
most RAC-2-expressing cells had normal morphology, some with abnormal
359
polarized growth and actin cytoskeleton disorganization were observed at a low
360
frequency (Figure 8F), which apparently did not significantly interfere with the
361
growth dynamics of RAC-2expressing yeast. SWAN-1-expressing cells showed
362
no apparent morphology and cytoskeletal defects (data not shown).
363
The observed defects in budding, polarized growth, and actin
364
disorganization mirrored those seen in selected mutants of the six yeast Rho
365
GTPases (Rho1-5p and Cdc42p) (DONG et al. 2003). Several yeast Rho proteins
366
are required to assemble the long actin filaments, which when absent lead to
367
polarity defects and actin patch disorganization; and Cdc42p is required for the
368
proper orientation of the long actin filaments. While yeast have no true Rac-
369
family GTPases, transgenic expression of CED-10 Rac, RAC-2 Rac, and MIG-2
18
370
Rac in yeast might ectopically interfere with the normal roles of the yeast Rho
371
GTPases, resulting in the cytoskeletal and cell polarity defects described above.
372
The growth defects caused by CED-10, RAC-2, and MIG-2 expression in
373
yeast were reduced when the yeast were co-transformed with SWAN-1.
374
Doubling time decreased in the SWAN-1 co-transformed strains (CED-10
375
expressing cells went from >10 hours to 4.5 hours, and MIG-2 cells went from 6
376
hours to 3.5 hours), and the percentages of budding cells increased (Figure 8A):
377
29% of CED-10-expressing cells were budding compared to 64% of CED-10
378
SWAN-1-expressing cells (p < 0.001). While the SWAN-1 co-transformed cells
379
still showed some cytoskeletal disorganization, it was not as severe as that seen
380
in CED-10 and MIG-2 alone, as indicated by increased percentages of budding
381
cells. These data indicate that SWAN-1 repressed CED-10 and MIG-2 activity in
382
yeast cells.
383
19
383
384
Discussion
Previous studies indicate that the UNC-115/abLIM actin-binding protein
385
controls lamellipodia and filopodia formation in response to Rac GTPase
386
signaling (STRUCKHOFF and LUNDQUIST 2003; YANG and LUNDQUIST 2005), but the
387
details of this pathway, including other molecules involved, remain unclear. A
388
two-hybrid screen with the UNC-115 LIM domains identified the novel SWAN-1
389
protein, the C. elegans member of a novel seven or eight -WD-repeat protein
390
family conserved in yeast, protozoa, plants, and animals. Subsequent directed
391
two-hybrid tests and coimmunoprecipitation experiments revealed that SWAN-1
392
interacted with both the UNC-115 LIM domains and each of the three C. elegans
393
Rac-family GTPases CED-10, RAC-2, and MIG-2. SWAN-1 might represent a
394
molecular link between Rac GTPases and UNC-115/abLIM. Functional assays
395
in C. elegans neurons, cultured mammalian 3T3 fibroblasts, and yeast indicate
396
that SWAN-1 is a new class of negative regulator of Rac GTPases.
397
Expression of activated Rac in C. elegans neurons leads to the formation
398
of ectopic lamellipodia and filopodia that is partially dependent on UNC-115
399
(STRUCKHOFF and LUNDQUIST 2003). Loss of swan-1 enhanced the effects of
400
CED-10 and RAC-2 activity, and transgenic expression of wild-type swan-1
401
repressed CED-10 and RAC-2 activity in neurons. In these transgenic assays,
402
no effect of SWAN-1 on MIG-2 activity was detected, despite the fact that SWAN-
403
1 and MIG-2 interacted strongly in the two-hybrid system. swan-1 loss of
404
function did enhance the effects of dominant mig-2 alleles on axon pathfinding,
405
suggesting that SWAN-1 also regulates MIG-2. SWAN-1 also repressed MIG-2
20
406
activity in yeast, consistent with the idea that SWAN-1 regulates all three Racs
407
CED-10, RAC-2, and MIG-2. Expression of C. elegans SWAN-1 also
408
suppressed the lamellipodia formed in 3T3 fibroblasts in response to human
409
Rac1 harboring the activating Q61L mutation, suggesting that the Rac-SWAN-1
410
interaction has been conserved in the evolutionary divergence of humans and C.
411
elegans.
412
swan-1 mutation also suppressed the cell corpse engulfment defects and
413
gonadal distal tip cell migration defects of the hypomorphic ced-10(n1993) allele,
414
indicating that SWAN-1 represses CED-10 activity in multiple developmental
415
processes. swan-1 did not suppress the distal tip cell migration defects of mig-
416
2(mu28), which is a predicted null allele of mig-2. ced-10(n1993) retains some
417
ced-10 activity, and this residual activity might be enhanced by loss of swan-1.
418
No residual MIG-2 activity is predicted to be present in the null mig-2(mu28)
419
mutant.
420
These experiments in C. elegans, mammalian fibroblasts, and yeast
421
support the idea that SWAN-1 inhibits Rac activity. swan-1 mutants have no
422
detectable phenotype on their own, and the effects of swan-1 in rac mutant
423
strains are generally weak albeit consistent across many different assays.
424
Generally, sensitized backgrounds were required to see an effect of swan-1 (e.g.
425
hypomorphic and dominant mutations in rac genes and transgenic expression
426
assays). SWAN-1 inhibited human Rac1 and to a lesser extent MYR::UNC-115
427
in fibroblasts. Possibly SWAN-1 is a direct inhibitor of UNC-115. However, no
428
effect of swan-1 on myr::unc-115 activity in C. elegans neurons was detected,
21
429
and SWAN-1 inhibited Rac activity in the yeast S. cerevisiae, which has no
430
molecule similar to UNC-115 encoded in its genome (GOFFEAU et al. 1996).
431
These data indicate that SWAN-1 is a direct inhibitor of Rac GTPase activity.
432
The inhibition of MYR::UNC-115 by SWAN-1 in fibroblasts might be an indirect
433
effect of Rac1 inhibition (i.e. MYR::UNC-115 might still require Rac activity for full
434
effect). However, it is possible that SWAN-1 is a direct inhibitor of both Rac and
435
MYR::UNC-115.
436
The effects of SWAN-1 on Rac activity are weak. The related SWAN-2
437
molecule showed no detectable functional overlap or similarity to SWAN-1,
438
suggesting that the two molecules have distinct functions. SWAN-1 might be a
439
fine-scale modulator that is involved in precisely tuning the amount of Rac
440
activity. Modest changes in the levels of Rac activity can have distinct
441
physiological outcomes. For example, in C. elegans, reducing the copy number
442
of rac genes can change an attractive semaphorin signal into an repulsive signal
443
(DALPE et al. 2004). In cultured mammalian fibroblasts, reduction of Rac1 activity
444
by as little as 30% leads a switch from random peripheral lamellipodia protrusion
445
to directed protrusion and directed cell migration (PANKOV et al. 2005). rac genes
446
display redundant functions in C. elegans and Drosophila whereby progressive
447
loss of rac gene activity leads to progressively more severe axon defects
448
(LUNDQUIST et al. 2001; NG et al. 2002). While there is clear overlap in rac gene
449
function, it is also possible that different levels of rac activity have distinct
450
physiological effects on axon development. SWAN-1 could also be a Rac
451
“fidelity factor” ensuring that background or transient Rac activation is
22
452
suppressed so that only strong, persistent Rac activity leads to directed
453
protrusion. swan-1(ok267) has no detectable phenotype on its own, but it is
454
possible that a subtle phenotype was not revealed in our endpoint phenotypic
455
analysis (e.g. the growth cones of swan-1 animals might behave differently than
456
those of wild-type, but they might eventually reach their targets).
457
GTPase activating proteins and guanine nucleotide dissociation inhibitors
458
are a well-characterized family of Rac negative regulators. GAPs inhibit small
459
GTPases by activating the GTPase activity of the molecules, favoring the
460
inactive, GDP-bound state, and GDIs prevent GTPase interaction with the
461
plasma membrane (MICHAELSON et al. 2001; VAN AELST and D'SOUZA-SCHOREY
462
1997). SWAN-1 does not resemble any known GAPs or GDIs, and, as expected,
463
SWAN-1 does not act as a GAP on human Rac1 (R. Steven, I. Blasutig, and A.
464
Pawson, personal communication). SWAN-1 interacted physically with both wild-
465
type and G12V-mutant activated Rac molecules, and swan-1 interacted
466
genetically with a variety of rac dominant mutations, including the activating
467
G12V and Q61L mutations, and the dominant mutations ced-10(n3246), ced-
468
10(n1993lq20), and mig-2(lq13), which affect Rac by unknown mechanisms.
469
SWAN-1 might bind to Racs and lock them in an inactive conformation, SWAN-1
470
might block sites of interaction with Rac effectors regardless of the GTP-bound
471
state of Rac, or SWAN-1 might regulate Rac interaction with the plasma
472
membrane.
473
474
UNC-115/abLIM and Racs act in the same pathway to promote protrusive
activity. SWAN-1 was isolated by its ability to interact with UNC-115 LIM
23
475
domains, and SWAN-1 is a negative regulator of Rac GTPases in the pathway.
476
UNC-115/abLIM might act as a scaffold for a complex that includes both
477
activators and inhibitors of protrusive activity. SWAN-1 might be recruited with
478
the Rac effector UNC-115/abLIM to Racs to precisely control levels of Rac
479
activity or to prevent spurious, transient Rac activity so that directed protrusion
480
can occur.
481
482
24
482
Materials and Methods
483
C. elegans culture and genetics. C. elegans were cultured using standard
484
techniques (EPSTEIN and SHAKES 1995). All experiments were performed at 20o
485
C. The swan-1(ok267) mutation was isolated by the C. elegans Gene Knockout
486
Consortium (kindly provided by G. Moulder and B. Barstead) and was outcrossed
487
to wild-type three times before phenotypic analysis. The homozygosity of swan-
488
1(ok267) in all outcrosses and single and double mutants was confirmed by
489
single-animal polymerase chain reaction (PCR) using primers that amplify a band
490
specific to the ok267 deletion. Germline transformation of C. elegans was
491
performed by standard techniques involving injection of a DNA mixture into the
492
syncytial germline of C. elegans hermaphrodites (EPSTEIN and SHAKES 1995).
493
RNA-mediated gene knockdown (RNAi) was performed by feeding nematode
494
strains E.coli expressing double-stranded RNA complementary to either swan-1
495
or swan-2 (KAMATH and AHRINGER 2003; TIMMONS et al. 2001).
496
Numbers of cell corpses in the region of the pharynx at the mid-three-fold
497
stage of embryogenesis (approximately 750 minutes post-fertilization) were
498
counted using differential interference contrast microscopy. Gonadal distal tip
499
cell migration defects were scored as the percentage of gonad arms that showed
500
defective morphology as previously described (LUNDQUIST et al. 2001). In Table
501
1, strains harboring a full-length swan-1::gfp transgene were scored. As a
502
control, non-transgene-bearing siblings from the same brood were also scored
503
and presented in Table 1.
25
504
Significance of differences of means in Table 1 were calculated using the
505
t-test, and significance of differences of proportions in Table 1 and Figures 4, 5,
506
7, and 8 were calculated using both the t-test and Fisher exact analysis.
507
Molecular biology. All fragments containing coding region that were generated
508
by PCR were sequenced to ensure that no mutations were introduced during the
509
procedure. The sequences of all PCR primers used in this work are available
510
upon request.
511
Microscopy. See Figure Legends for details about each micrograph. Unless
512
otherwise noted, all images were captured on a Leica DMRE microscope with a
513
40x Planapo objective and 10x magnifier using a Hamamatsu Orca C4742-94
514
camera and Openlab software. Images were processed in Photoshop.
515
UNC-115 LIM domain two-hybrid screen. Vectors for the two-hybrid system
516
were kindly provided by S. Elledge. The coding region for the UNC-115 LIM
517
domains was amplified by PCR and cloned in-frame to the GAL4 DNA-binding
518
domain in the vector pAS1-CYH (the “bait” construct). The yeast strain Y190
519
was transformed with the bait construct by standard techniques, and
520
autoactivation of the bait construct was tested by growth on 25mM 3-
521
aminotriazole (3-AT) and X-Gal, which assess expression of HIS5 and LacZ
522
reporter gene expression in Y190. The bait construct showed no autoactivation:
523
it did not allow Y190 yeast to grow on 3-AT (no HIS5 expression), nor did result
524
in blue colonies when transgenic yeast were grown on X-Gal (no LacZ
525
expression).
26
526
Y190-bait transgenic yeast were transformed with a C. elegans random-
527
primed cDNA library cloned into the pACT vector that harbors the GAL4
528
activation domain (the library was kindly provided by R. Barstead). Yeast were
529
plated at high density on medium containing 25mM 3-AT and X-Gal, and colonies
530
that were blue (indicating HIS5 expression and LacZ expression) were selected
531
for further analysis. From an estimated 2 million cDNAs screened, two cDNAs
532
representing the swan-1 locus were recovered. These two cDNAs did not result
533
in autoactivation when present in Y190 without the bait plasmid, indicating that
534
both plasmids are needed for activation.
535
The coding regions of the three C. elegans rac genes ced-10, mig-2, and
536
rac-2, and the non-LIM-domain-containing portion of unc-115 were generated by
537
PCR and fused in frame to the GAL4 DNA-binding domain in the pAS1-CYH
538
plasmid. None of these constructs resulted in autoactivation. These constructs
539
were then used in direct two-hybrid tests with the pACT::SWAN-1 plasmid.
540
Coimmunoprecipitation experiments. Hela cells and HEK293 cells were
541
grown to approximately 80% confluency were transfected using Fugene6
542
(Roche, Indianapolis, IN). The full-length unc-115 coding region was generated
543
by PCR and cloned in-frame to enhanced green fluorescent protein (EGFP) in
544
the pEGFP1 vector (Clontech, Mountain View, CA). The swan-1 coding region
545
was cloned in frame to both the Myc epitope (MYC::SWAN-1) and the 3xFLAG
546
epitope (FLAG::SWAN-1) in the pCMV-Myc and pCMV-3xFLAG vectors,
547
respectively (Clontech, Mountain View, CA). MYC::RAC-2 and FLAG::RAC-2
548
plasmids were similarly constructed. In most cases, 16µg of plasmid was used in
27
549
single transfections and 8µg of each plasmid were used in double transfections in
550
75 cm2 flasks. For RAC-2 plasmids, 8µg were used because higher
551
concentrations led to cell inviability. After 48 hr of growth after transfection, cells
552
were washed with ice-cold PBS and 1 ml of ice-cold lysis buffer (150mM
553
NaCl/10mM Tris (pH7.4)/1mM EDTA/1mM EGTA/ 0.5% NP-40/ 0.2mM PMSF/
554
1% protease inhibitor cocktail (Sigma, Saint Louis, MO)) was added. Cells were
555
scraped from the bottom of the flasks, incubated for 1 hr at 4º C with shaking,
556
and centrifuged for 15 min at 16,000 x g in a microcentrifuge at 4ºC.
557
Supernatants were precleared by incubating with 80µl of pre-equilibrated protein
558
G SepharoseTM bead slurry (Amersham Pharmacia Biotech, Piscataway, NJ) for
559
2 hr at 4ºC.
560
An 80-µl bead slurry in 1ml ice-cold lysis buffer was preloaded with anti-
561
Myc monoclonal antibody (BD Biosciences, Palo Alto, CA ) or anti-FLAG M2
562
monoclonal antibody (Sigma, St. Louis, MO). After 4 hr, the beads were washed
563
four times with lysis buffer and incubated with the precleaned lysates at 4°C for
564
12 hr. Beads were washed in lysis buffer four times and the immune complexes
565
were eluted from the beads by boiling in 40µl 2xSDS loading buffer with 1mM
566
DTT for 5 min. The supernatants were analyzed by sodium dodecyl sulfate-
567
polyacrylamide gel electrophoresis (SDS-PAGE) and western blots using anti-
568
Myc, anti-FLAG or anti-UNC-115 antibodies (see below) and HRP-labeled
569
secondary antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD)
570
with chemilumiscent autoradiography (Pierce, Rockford, IL).
28
571
Figure 1C and D show coimmunoprecipitation of UNC-115 and SWAN-1
572
and RAC-2 and SWAN-1. For each coimmunoprecipitation experiment, the
573
western blot was also probed with the antibody used for immunoprecipitation to
574
ensure that the immunoprecipitation procedure was robust (Figure 1).
575
Generation of an UNC-115 antiserum. The coding region for UNC-115
576
residues 154-527 was inserted into the pQE9 vector (Qiagen, Valencia, CA) in
577
frame with the RGS-6-histidine epitope, and fusion protein was produced by
578
bacterial expression. SDS-PAGE gel fragments containing the fusion protein
579
were used as an antigen to immunize two rabbits (Caltag Laboratories,
580
Healdsburg, CA). On western blots, serum from the immunized rabbits but not
581
pre-immune serum recognized a bacterially-expressed glutathione-S-transferase
582
(GST) fusion with same portion of UNC-115 (data not shown) (residues 154-527;
583
produced by inserting the unc-115 fragment into the pGEX4T vector (Amersham
584
Pharmacia Biotech, Piscataway, NJ)). UNC-115-specific antibodies were purified
585
from the antiserum by their affinity for the GST::UNC-115 fusion protein using
586
glutathione sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ).
587
This affinity-purified polyclonal antibody recognized a single band of
588
approximately 70 kD in western blots of lysates of C. elegans (the estimated size
589
of full-length UNC-115 polypeptide is 72kD) (data not shown).
590
swan-1 expression analysis. The upstream region of swan-1 excluding the
591
initiator ATG codon (3412-7549 relative to cosmid F53C11) was amplified,
592
inserted into the pPD95.77 GFP expression vector (kindly provided by A. Fire),
593
and used to drive the expression of GFP in transgenic animals. A full-length
29
594
fusion of the swan-1 coding region to gfp was constructed by amplifying the
595
swan-1 upstream region and entire coding region (3412-9941 relative to cosmid
596
F53C11) from genomic DNA and fusing this fragment in frame to gfp such that
597
the transgene was predicted to encode a full-length SWAN-1 protein with GFP at
598
the C-terminus. swan-1 expression constructs were used at 5ng/µl in
599
transformation experiments.
600
SWAN-1 transgenic assays in C. elegans. For the loss-of-function swan-
601
1(ok267) experiments, previously-described constitutively-active rac(G12V)
602
transgenes were used (STRUCKHOFF and LUNDQUIST 2003). PDE morphological
603
defects were scored as previously described (STRUCKHOFF and LUNDQUIST 2003):
604
for each genotype, the percentages of PDE neurons exhibiting ectopic neurites
605
and ectopic lamellipodia and filopodia were determined. After construction of
606
each transgene in the swan-1(ok267) background, the transgene was crossed
607
away from swan-1(ok267) and axon defects were scored again to ensure that the
608
transgene caused defects at percentages similar to before the experiment began.
609
This ensured that the transgenes had not been altered during the course of the
610
experiment and the phenotypic change was due to the presence of swan-
611
1(ok267).
612
For the swan-1(+) expression experiments, new transgenes were
613
constructed that contained a plasmid harboring the swan-1(+) genomic region
614
(bases 3412-9972 inclusive of cosmid F53C11), a rac(G12V) plasmid, or both. In
615
all cases an osm-6::gfp plasmid was included as a neuronal marker to visualize
616
the PDE neuron. When constructing transgenic strains, rac(G12V) plasmids
30
617
were used at 1ng/µl and other plasmids were used at 5ng/µl. Multiple transgenic
618
lines were generated for each experiment to ensure uniformity of results, and
619
results from each line were accumulated to derive a percentage of PDE axon
620
defects. In all C. elegans experiments, at least 100 animals were scored for
621
each genotype. Significance of differences in proportions were calculated using
622
the t-test.
623
SWAN-1 transgenic assays in fibroblasts. Mammalian NIH 3T3 fibroblasts
624
were grown and transformed as previously described (YANG and LUNDQUIST
625
2005). Briefly, cells grown in six-well plates to approximately 50% confluency on
626
polylysine-coated cover slips were transfected by addition of a mixture of the
627
transgene DNA and the Fugene reagent. Cells were transfected with
628
combinations of 1µg of each of the MYC::SWR-1 construct, a MYR::UNC-115
629
construct, and a CFP::RAC-1(Q61L) construct, which encodes an activated
630
version of human Rac1 fused to cyan fluorescent protein (CFP) (kindly provided
631
by T. Meyer). Cells were co-transfected with egfp to identify transfected cells,
632
and cells transfected with egfp alone were used as a control. After transfection,
633
cells were allowed to grow for 6 hours and deprived of serum (serum-containing
634
medium was replaced by DMEM). Cells were grown for another 12 hours and
635
fixed in 3.7% paraformaldehyde. The fixed cells were treated with 1 µg/ml of
636
rhodamine-labeled phalloidin to visualize the actin cytoskeleton. Cells were
637
mounted in 50% glycerine in PBS for epifluorescence microscopy. For each
638
experiment, at least three transfections were performed and >100 cells were
639
scored to ensure consistency of results.
31
640
SWAN-1 transgenic assays in yeast. All yeast were grown at 30oC. For
641
growth curve analysis, Y190 yeast strains harboring different plasmid constructs
642
were grown in selective medium and diluted to approximately 0.1 OD600 units in
643
YPD medium. Growth rates were monitored by sampling the OD600 culture
644
densities each hour for eight hours (see Figure 8). Each strain was tested at
645
least twice, and standard deviations were indicated by error bars in Figure 9. To
646
visualize the yeast actin cytoskeleton with rhodamine-labeled phalloidin, cells
647
were grown in selective medium, diluted to approximately 0.1 OD600 units in YPD,
648
and grown to mid-log phase (0.3-0.7 OD600) (cells harboring the CED-10 plasmid
649
were assayed after 8 hr). Cells were fixed for 1 hr in 20% formaldehyde in 0.5M
650
potassium phosphate pH 6.5 and overnight in 4% formaldehyde in 100mM
651
potassium phosphate pH 6.5. Cells were washed in PBS and permeabilized in
652
0.2% Triton X-100 in PBS for 15 min, washed three times in PBS, incubated with
653
rhodamine-phalloidin (1µg/ml) and 4’,6-diamidino-2-phenylindole dihydrochloride
654
(DAPI) (1µg/ml) in PBS for 1 hr in the dark, washed three times in PBS, and
655
mounted for fluorescence microscopy in PBS with 90% glycerol and 1mg/ml 1,4-
656
diazabicyclo[2.2.2]octane (DABCO) antifade reagent.
657
658
32
658
Acknowledgments
659
We thank E. Struckhoff for technical assistance, R. Barstead, G. Moulder, and
660
the C. elegans Gene Knockout Consortium for C. elegans strains, R. Barstead
661
for providing the two-hybrid cDNA library, T. Meyer for providing the Rac1(Q61L)
662
clone, K. Neufeld and T.C. Gamblin for reagents and technical assistance, and
663
the Caenorhabditis Genetics Center, sponsored by the National Center for
664
Research Resources, for providing C. elegans strains. This work was supported
665
by NIH grant NS40945 and NSF grant IBN93192 to E.A.L., and NIH grant P20
666
RR016475 from the INBRE Program of the National Center for Research
667
Resources.
668
669
33
669
Figure Legends
670
Figure 1. SWAN-1 interacts with the UNC-115 LIM domains and with Rac
671
GTPases in a two-hybrid system. (A) A diagram of the 639-residue UNC-115
672
polypeptide. Indicated are the regions used in the two-hybrid screen and to
673
generate an anti-UNC-115 polyclonal antiserum. (B) Yeast harboring different
674
bait plasmids (across the top) without and with the SWAN-1 prey plasmid (see
675
Materials and methods). Yeast were grown on medium containing X-Gal to
676
indicate lacZ activity and a positive interaction (blue). Yeast harboring the bait
677
plasmids or SWAN-1 alone were overgrown to insure absence of lacZ activity.
678
Images were captured by an Epson flatbed scanner and processed in
679
Photoshop. (C) and (D) SWAN-1 coimmunoprecipitated with UNC-115 and
680
RAC-2 in HeLa cell lysates. (C) A western blot using anti-UNC-115 antiserum.
681
Lysates were prepared from cells expressing combinations of MYC::SWAN-1
682
and UNC-115::EGFP. The 100-kD UNC-115::EGFP band is indicated, as is the
683
Ig heavy chain of the anti-Myc antibody (50 kD), which cross-reacts with the
684
secondary antibody. UNC-115::EGFP was coimmunoprecipitated only when
685
MYC::SWAN-1 was present. The successful immunoprecipitation of
686
MYC::SWAN-1 was confirmed by western blot with an anti-Myc antibody (data
687
not shown). (D) MYC::RAC-2 coimmunoprecipitated with FLAG::SWAN-1.
688
Shown are western blots from two immunoprecipitation experiments. The left
689
panels 1 and 3 are the results of a MYC::RAC-2 immunoprecipitation, and right
690
panels 2 and 4 are the results of a FLAG::SWAN-1 immunoprecipitation. Across
691
the top is indicated the transgenes expressed in cells for each experiment. The
34
692
top two panels (1 and 2) show that FLAG::SWAN-1 co-immunoprecipitated with
693
MYC::RAC-2 and that MYC::RAC-2 co-immunoprecipitated with FLAG::SWAN-1,
694
respectively. The Ig heavy chain from the anti-FLAG antibody (50kD), also
695
detected by the secondary antibody, and FLAG-SWAN-1 (45 kD) were similar in
696
size but could be distinguished (panel 1). The Ig light chain from the anti-Myc
697
antibody (25kD) and MYC::RAC-2 (20kD) were more difficult to resolve (panels 2
698
and 3). In three independent experiments, MYC::RAC-2 was evident as a
699
thickening of the band below the Ig light chain and by the smaller bands (possible
700
degradation products of MYC::RAC-2) below 23 kD (asterisk). The smaller
701
products always correlated with the presence of MYC-RAC-2. Panels 3 and 4
702
are the controls for immunoprecipitation (MYC::RAC-2 was immunoprecipitated
703
with anti-Myc antibody (panel 3); and FLAG::SWAN-1 was immunoprecipitated
704
by anti-FLAG antibody (panel 4)). FLAG::SWAN-1 co-immunoprecipitated only in
705
the presence of MYC::RAC-2; and MYC::RAC-2 co-immunoprecipitated only in
706
the presence of FLAG::SWAN-1.
707
708
Figure 2. The swan-1 locus. (A) A depiction of the swan-1 and swan-2 loci. 5’
709
is to the left. Boxes represent exons; black boxes represent coding regions. SL1
710
indicates where a the trans-spliced leader sequence SL1 is attached to the 5’
711
end of the swan-1 cDNA; and the site of a poly-A tail on the swan-1 cDNA is
712
shown. The extent of the swan-1(ok267) deletion is indicated. (B) The swan-1
713
coding region. Upper-case letters represent exons present in three independent
714
cDNAs that were sequenced, and bold letters represent the open reading frame.
35
715
Lower case indicates intron sequences inferred from the sequence for the
716
F53C11.8 region on Wormbase. Underlined sequences represent the extent of
717
the ok267 deletion. Base 1 of the sequence is where an SL1 trans-spliced leader
718
sequence is present in a cDNA generate by 5’ RACE. The two arrows represent
719
the start points of two independent cDNAs isolated in the yeast two-hybrid
720
screen. The sequences of these two cDNAs as well as those of two other
721
independently-derived cDNAs yk326a5 and yk343g2 (kindly provided by Y.
722
Kohara) were determined and were identical throughout the open reading frames
723
of the cDNAs. The end of the sequence is where a poly-A tail is present in
724
yk1174f05 (data from Wormbase).
725
726
Figure 3. The SWAN-1 polypeptide. (A) The amino acid sequence of the 388-
727
residue SWAN-1 polypeptide. Residues whose coding region was removed by
728
the ok267 deletion are underlined. (B) Schematic structure of the SWAN-1
729
polypeptide. Regions with similarity to the WD repeat domain are indicated, as is
730
the C-terminal conserved domain (CTD) unique to SWAN-1-family polypeptides.
731
The region of the molecule whose coding region was removed by ok267 is
732
indicated. (C) The SWAN-1-family is conserved in animals, plants, fungi, and
733
protozoa. Predicted amino acid sequences were aligned by ClustalW: Ce, C.
734
elegans SWAN-1 (Genbank accession number NP_506418) and SWAN-2
735
(NP_506417); Dm, Drosophila melanogaster (NP_608461); Dr, Danio rerio
736
(zebrafish) (NP_956363); Hs, Homo sapiens (AAH01264); AN11 from Petunia x
737
hybrida (AAC18914); At, Arabidopsis thaliana (NP_172751); Sp,
36
738
Schizosaccharomyces pombe (CAA21079); Sc, Saccharomyces cerevisiae
739
(NP_015077); Dd, Dictyostelium discoideum (protozoan slime mold)
740
(XP_643620). Residues identical in a majority of the molecules are highlighted in
741
dark grey. Regions similar to the WD repeat (WD1-8) and the conserved C-
742
terminal domain (CTD) are boxed in light grey. Members from the yeast S.
743
cerevisiae and S. pombe include additional amino acid residues, not found in
744
other members, between WD5 and WD6, WD7 and WD8, and in WD8. The
745
SWAN-1-like molecule from the protozoan D. discoideum contains a run of 38
746
consecutive serines in the region that corresponds to WD2.
747
748
Figure 4. SWAN-1 negatively modulates activated Rac. (A) and (B) are
749
fluorescence micrographs of the PDE neurons of young adult animals with gfp
750
expression driven by the osm-6 promoter in the PDE neuron. Anterior is left and
751
dorsal is up; scale bar in A = 5µm. (A) Wild-type PDE morphology. A ciliated
752
dendrite extends dorsally, and a single unbranched axon extends ventrally to the
753
ventral nerve cord (out of focus; dashed line). (B) A PDE neuron with CED-
754
10(G12V) expression. The neuron displayed ectopic protrusive structures that
755
resemble lamellipodia and filopodia. Ectopic axons and axon branches were
756
also observed (data not shown). (C) Loss-of-function swan-1(ok267) enhances
757
the effects of activated CED-10(G12V) and RAC-2(G12V) transgenes.
758
Genotypes analyzed define the X axis, and the percent of PDE neurons
759
displaying ectopic lamellipodia/filopodia and ectopic neurites define the Y axis.
760
Error bars represent the standard error of the proportion. (D) Transgenic
37
761
expression of wild-type swan-1 suppresses transgenic CED-10(G12V) and RAC-
762
2(G12V). The X axis is compositions of transgenes analyzed. Transgenes
763
included activated rac(G12V) constructs with and without wild-type swan-1 DNA
764
(see Materials and methods). Multiple transgenic lines (n) were analyzed for
765
each combination. The Y axis and error bars are as described in (C).
766
As determined by a t-test and by Fisher exact analysis, the effects of
767
swan-1(ok267), swan-1(RNAi), and swan-1(+) expression on CED-10(G12V)
768
and RAC-2(G12V) were significant (p < 0.001).
769
770
Figure 5. swan-1(ok267) enhances dominant ced-10 and mig-2 alleles. (A) and
771
(B) are fluorescence micrographs of osm-6::gfp expression in PDE neurons from
772
ced-10(n3246); swan-1(ok267) animals. Anterior is left and dorsal is up; scale
773
bar in A = 5µm. Dashed lines indicate the dorsal and ventral limits of the body of
774
the animal. (A) demonstrates a mild PDE axon pathfinding defect, where the
775
axon is misguided but reaches the ventral nerve cord (out of focus; dashed line)
776
in the vicinity of the cell body. (B) represents a severe PDE axon pathfinding
777
defect, where the axon fails to reach the ventral nerve cord. (C) Genotypes
778
analyzed define the X axis, and the Y axis represents the percent of PDE
779
neurons in each genotype displaying wild-type morphology (white), mild axon
780
pathfinding defects (grey), and severe axon pathfinding defects (black). White
781
error bars represent the standard error of the proportion of severe axon defects,
782
and black error bars represent standard error of the proportion of combined mild
783
and severe defects. Differences in the severe category between the activated
38
784
rac mutation alone and the double with swan-1(ok267) were significant (p <
785
0.001; t-test and Fisher exact analysis).
786
787
Figure 6. swan-1::gfp is expressed in all cells except the intestine. Micrographs
788
of an embryo, approximately 270 minutes after fertilization, harboring the full-
789
length swan-1::gfp fusion transgene are shown (see Materials and methods). An
790
HCX Planapo 63x objective (1.3 n.a.) and a 10x magnifier were used. The
791
dashed line surrounds the intestinal precursor cells. Scale bar in A is 10µm. (A)
792
is a fluorescence micrograph; (B) is a differential interference contrast
793
micrograph; and (C) is a merged image.
794
795
Figure 7. SWAN-1 inhibits human Rac1 activity in cultured cells. (A) A serum-
796
starved NIH 3T3 fibroblast transfected with activated human Rac1(Q61L), fixed
797
and stained with rhodamine-phalloidin to visualize the actin cytoskeleton. The
798
arrow indicates a lamellipodial ruffle surrounding the cell. (B) The combinations
799
of transgenes used to transfect serum-starved fibroblasts define the X axis.
800
EGFP was included in each mix. Rac1 harbored the Q61L mutation, and SWAN-
801
1 was tagged with the Myc epitope. The percent of transfected cells (as judged
802
by the EGFP expression) displaying lamellipodia define the Y axis. Error bars
803
represent standard error of the proportion. The effects of SWAN-1 expression on
804
Rac1(G12V) activity was significant (p < 0.01) as judged by a t-test and Fisher
805
exact analysis. (C) A fluorescence micrograph of a serum-starved fibroblast
806
harboring a MYR::UNC-115::EGFP transgene. Fluorescence is from MYR::UNC-
39
807
115::EGFP. The arrow indicates a large lamellipodial protrusion with multiple
808
filopodia. (D) is as described in (B). The effects of SWAN-1 expression on
809
MYR::UNC-115 activity were not significant as judged by a t-test (p = 0.1076)
810
and Fisher exact analysis. Scale bar in A = 5µm.
811
812
Figure 8. SWAN-1 inhibits C. elegans Rac activity in yeast. (A) A growth curve
813
for yeast harboring different combinations of transgenes (X axis is timepoint; Y
814
axis is OD600) (see Materials and Methods). Y190 is the background strain;
815
CED-10 and MIG-2 are fusions of the molecules with the DNA-binding domain of
816
GAL4 in the pAS1CYH two-hybrid vector; and SWAN-1 is a fusion of the
817
molecule to the activation domain of GAL4 in the pACT two-hybrid vector. Error
818
bars represent the standard deviation of two experiments. (B) Rac expression in
819
yeast perturbs polarized cell division. Six hours after dilution to 0.1 OD600, yeast
820
cells harboring different transgenes (X axis), cells were fixed and stained with
821
rhodamine-phalloidin and DAPI. On the Y axis is the percent of cells from each
822
strain in the process of polarized cell budding. Error bars represent standard
823
error of the proportion. (C-G) Fluorescence micrographs of yeast cells from
824
different strains stained with rhodamine-phalloidin to visualize actin (actin) and
825
DAPI to visualize DNA in the nucleus (DAPI). In (C) arrows point to polarized
826
actin cables in the mother cell and arrowheads indicate the actin patches in the
827
daughter cell. In (D), the arrow points to a disorganized actin cable in a mother
828
cell. In (D), (E), and (F), arrowheads indicate actin patches ectopically present in
40
829
mother cells. (G) shows a large, unpolarized cell undergoing non-polarized
830
growth without cell division. Scale bar in A = 10µm.
41
References
ADAMS, A. E., and J. R. PRINGLE, 1984 Relationship of actin and tubulin distribution to
bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J
Cell Biol 98: 934-945.
DALPE, G., L. W. ZHANG, H. ZHENG and J. G. CULOTTI, 2004 Conversion of cell
movement responses to Semaphorin-1 and Plexin-1 from attraction to repulsion
by lowered levels of specific RAC GTPases in C. elegans. Development 131:
2073-2088.
DE VETTEN, N., QUATTROCCHIO, F., MOL, J., AND KOES, R., 1997 The an11 locus
controlling flower pigmentation in petunia encodes a novel WD-repeat protein
conserved in yeast, plants and animals. Genes Dev. 11: 1422-1434.
DICKSON, B. J., 2001 Rho GTPases in growth cone guidance. Curr Opin Neurobiol 11:
103-110.
DICKSON, B. J., 2002 Molecular mechanisms of axon guidance. Science 298: 1959-1964.
DONG, Y., D. PRUYNE and A. BRETSCHER, 2003 Formin-dependent actin assembly is
regulated by distinct modes of Rho signaling in yeast. J Cell Biol 161: 1081-1092.
EPSTEIN, H. F., and D. C. SHAKES, 1995 Caenorhabditis elegans: Modern Biological
Analysis of an Organism. Academic Press, New York.
GOFFEAU, A., B. G. BARRELL, H. BUSSEY, R. W. DAVIS, B. DUJON et al., 1996 Life with
6000 genes. Science 274: 546, 563-547.
HALL, A., 1998 Rho GTPases and the actin cytoskeleton. Science 279: 509-514.
KAMATH, R. S., and J. AHRINGER, 2003 Genome-wide RNAi screening in Caenorhabditis
elegans. Methods 30: 313-321.
KISHORE, R. S., and M. V. SUNDARAM, 2002 ced-10 Rac and mig-2 function redundantly
and act with unc-73 trio to control the orientation of vulval cell divisions and
migrations in Caenorhabditis elegans. Dev Biol 241: 339-348.
LUNDQUIST, E. A., 2003 Rac proteins and the control of axon development. Curr Opin
Neurobiol 13: 384-390.
LUNDQUIST, E. A., R. K. HERMAN, J. E. SHAW and C. I. BARGMANN, 1998 UNC-115, a
conserved protein with predicted LIM and actin-binding domains, mediates axon
guidance in C. elegans. Neuron 21: 385-392.
LUNDQUIST, E. A., P. W. REDDIEN, E. HARTWIEG, H. R. HORVITZ and C. I. BARGMANN,
2001 Three C. elegans Rac proteins and several alternative Rac regulators control
axon guidance, cell migration and apoptotic cell phagocytosis. Development 128:
4475-4488.
MICHAELSON, D., J. SILLETTI, G. MURPHY, P. D'EUSTACHIO, M. RUSH et al., 2001
Differential localization of Rho GTPases in live cells: regulation by hypervariable
regions and RhoGDI binding. J Cell Biol 152: 111-126.
NEER, E. J., C. J. SCHMIDT, R. NAMBUDRIPAD and T. F. SMITH, 1994 The ancient
regulatory-protein family of WD-repeat proteins. Nature 371: 297-300.
NG, J., T. NARDINE, M. HARMS, J. TZU, A. GOLDSTEIN et al., 2002 Rac GTPases control
axon growth, guidance and branching. Nature 416: 442-447.
42
OTIENO, C. J., J. BASTIAANSEN, A. M. RAMOS and M. F. ROTHSCHILD, 2005 Mapping
and association studies of diabetes related genes in the pig. Anim Genet 36: 3642.
PANKOV, R., Y. ENDO, S. EVEN-RAM, M. ARAKI, K. CLARK et al., 2005 A Rac switch
regulates random versus directionally persistent cell migration. J Cell Biol 170:
793-802.
RAFTOPOULOU, M., and A. HALL, 2004 Cell migration: Rho GTPases lead the way. Dev
Biol 265: 23-32.
REDDIEN, P. W., and H. R. HORVITZ, 2000 CED-2/CrkII and CED-10/Rac control
phagocytosis and cell migration in Caenorhabditis elegans. Nat Cell Biol 2: 131136.
SHAKIR, M. A., J. S. GILL and E. A. LUNDQUIST, 2006 Interactions of UNC-34 Enabled
with Rac GTPases and the NIK kinase MIG-15 in Caenorhabditis elegans axon
pathfinding and neuronal migration. Genetics 172: 893-913.
SMITH, T. F., C. GAITATZES, K. SAXENA and E. J. NEER, 1999 The WD repeat: a
common architecture for diverse functions. Trends Biochem Sci 24: 181-185.
SONDEK, J., A. BOHM, D. G. LAMBRIGHT, H. E. HAMM and P. B. SIGLER, 1996 Crystal
structure of a G-protein beta gamma dimer at 2.1A resolution. Nature 379: 369374.
SOTO, M. C., H. QADOTA, K. KASUYA, M. INOUE, D. TSUBOI et al., 2002 The GEX-2 and
GEX-3 proteins are required for tissue morphogenesis and cell migrations in C.
elegans. Genes Dev 16: 620-632.
STEVEN, R., T. J. KUBISESKI, H. ZHENG, S. KULKARNI, J. MANCILLAS et al., 1998 UNC73 activates the Rac GTPase and is required for cell and growth cone migrations
in C. elegans. Cell 92: 785-795.
STRUCKHOFF, E. C., and E. A. LUNDQUIST, 2003 The actin-binding protein UNC-115 is
an effector of Rac signaling during axon pathfinding in C. elegans. Development
130: 693-704.
TESSIER-LAVIGNE, M., and C. S. GOODMAN, 1996 The molecular biology of axon
guidance. Science 274: 1123-1133.
TIMMONS, L., D. L. COURT and A. FIRE, 2001 Ingestion of bacterially expressed dsRNAs
can produce specific and potent genetic interference in Caenorhabditis elegans.
Gene 263: 103-112.
VAN AELST, L., and C. D'SOUZA-SCHOREY, 1997 Rho GTPases and signaling networks.
Genes Dev 11: 2295-2322.
VICENTE-MANZANARES, M., D. J. WEBB and A. R. HORWITZ, 2005 Cell migration at a
glance. J Cell Sci 118: 4917-4919.
WALKER, A. R., P. A. DAVISON, A. C. BOLOGNESI-WINFIELD, C. M. JAMES, N.
SRINIVASAN et al., 1999 The TRANSPARENT TESTA GLABRA1 locus, which
regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,
encodes a WD40 repeat protein. Plant Cell 11: 1337-1350.
WHITE, J. G., E. SOUTHGATE, J. N. THOMSON and S. BRENNER, 1986 The structure of the
nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc.
Lond. 314: 1-340.
43
YANG, Y., and E. A. LUNDQUIST, 2005 The actin-binding protein UNC-115/abLIM
controls formation of lamellipodia and filopodia and neuronal morphogenesis in
Caenorhabditis elegans. Mol Cell Biol 25: 5158-5170.
YU, T. W., and C. I. BARGMANN, 2001 Dynamic regulation of axon guidance. Nat
Neurosci 4 Suppl: 1169-1176.
ZIPKIN, I. D., R. M. KINDT and C. J. KENYON, 1997 Role of a new Rho family member in
cell migration and axon guidance in C. elegans. Cell 90: 883-894.
44
Table 1. swan-1 Loss-of-Function Suppresses ced-10(n1993) but not
mig-2(mu28).
Number of
persistent cell
corpses/embryo1
Percent Distal
Tip Cell
Migration
Defects2
+
0.05±0.3
n=50
0
n>200
swan-1(ok267)
0.1±0.3
n=56
3±1
n=132
ced-10(n1993)
14.9±3.84
n=97
24±46
n=120
ced-10(n1993); swan1(ok267)
9.6±4.24
n=89
7±26
n=113
ced-10(n1993); swan1(ok267); Ex[swan-1(+)]3
15.6±3.45
n=119
29±37
n=191
ced-10(n1993); swan1(ok267) (nontransgene-bearing sibs)3
11.7±3.65
n=115
12±27
n=174
mig-2(mu28)
NA
16±3
n=145
mig-2(mu28); swan1(ok267)
NA
17±4
n=101
Genotype
1
Persistent corpses in the region of the pharynx were scored in three-fold larvae
prior to hatching (~750 minutes post-fertilization). (± standard deviation).
2
In young adult hermaphrodites, distal tip cell migration was scored as defective
if the gonad arm failed to execute its complete migration or if the gonad arm was
misguided. (± standard error of the proportion).
45
3
Ex[swan-1(+)] is a transgenic array harboring wild-type swan-1. non-transgenebearing sibs are the siblings of transgenic animals from the same brood that did
not inherit the transgene.
4,5
Distributions are significantly different (t-test, p < 0.001).
6,7
Proportions are significantly different (t-test and Fisher exact analysis, p <
0.001).
46
LIM
LIM
LIM
A
B
UNC-115 abLIM
VHD 639 aa
two-hybrid bait
+ SWAN-1
antigen
Figure 1
- SWAN-1
SWAN-1
alone
UNC-115
LIM
CED-10
RAC-2
MIG-2
C
Myc::SWAN-1 IP
MYC::SWAN-1
UNC-115::EGFP
UNC-115::EGFP
100 kD
Ig heavy chain
50 kd
anti-UNC-115 western
D
MYC::RAC-2 IP
FLAG::SWAN-1 IP
MYC::RAC-2
FLAG::SWAN-1
Ig heavy chain
1
50 kD
FLAG::SWAN-1
45 kD
Ig light chain
25 kD
MYC::RAC-2
23 kD
anti-FLAG western
anti-Myc western
3
anti-Myc western
*
MYC::RAC-2
23 kD
4
Ig heavy chain
50 kD
*
anti-FLAG western
Figure 1
Ig light chain
25 kD
2
FLAG::SWAN-1
45 kD
A
SL1
1kb
ok267 deletion
Poly-A
swan-1 (F53C11.8)
swan-2 (F53C11.7)
B
AAACTTGTGAACCCTTATCACCTGCAATGGCTACCAATGGTCACGCAGTG
AACGGAGAAGCTCATGTTATGCAACAAGTCGCACAACAACCAGCTCCACC
AGGACCATTAGCTCCGCAAGGGATCGGAAGACAGAACGAAATTGTGATCG
GAGGACGTGATCGACGTTGTGAGATCTATAAATTCACGTCTGATCAACAG
TTGTATGCATCGGCATGGAGCAACAAAAATGATATCAAATTTCGACTTGC
TGTGGGAACAGTATCCGACGTGTCCGTTAACCCATGTGCCGCAAACAAGg
tgagtttttgaaaaatttgaaaacgtgtgagtggtaatgttggccatagt
Caataaggtcccatttactgaaataccacaaaaaacgcaattctaaaaaa
cgggtttttgcaaattctaatctactgtttaagttgtctacaaaataaga
actgcaagattaaacagcgatcgttcttatttttgagttgagttatattt
ttgagtcgagttttactatagttcaaagacacaatttagaaaaactggaa
Atcggaacaaatcgaaattgaactgttcaggttttaaacataatttttgt
ctttccttttgcggtatttcagtatgtatattaaaacatttcgaatattt
tgaattgaaaaccctatagactgtactccaatgtaaaagttaaaaaaaaa
aaaaacgaaatttatatgtattttgaaacataacagttccaattaaaacg
ttttcagGTATCAATTGTACAACTAAAAGACGAAACTGGCGAGCTTGTCG
AGACGGCGAGCTTTCCAATGGAGTTCCCAGCCAATGCAGTCGGTTTTATT
CCGGATCCAGACAACGTTTACCCTGATCTCATTGCCACAACATCAGATTG
CCTTCGCCTCTGGCGAGTAGTTGATGGAAAAGTTCATCCAGACGCAGTTA
TGATCAATAATACTgtacgttaaacctgtaaatttttagtgttcaatgga
attttcagAACTCACAATATGGATCAGCTTTGACAAGTTTCGACTGGAAC
GAGCTGGAGCCGAGGTATATTGGAGTTTCCAGTGTAGATACGACTTGCAC
AATTTATGATGTTGAGgtgagttataattttgataagatttagtgaaatg
tttacaaatattcagGTTGGTTGCGCAATTGGGCAGACCAAGCCGACAGC
TCCATTCACACTCAAAACACAACTGATTGCTCATGACAAGCCTGTTCACG
ATATCGAATTTGCTAAAATCAATGGCGGTCGTGATCATTTTGCAACTGTT
Ggtgagtcttgtctagctagtatttgcatctttttataaacattacgagt
ttccagGAGCTGACGGAAGTGCCCGAATGTTTGATCTTCGACATTTAAAC
CACTCGACAATTGTCTATGAAGATCCAAACAAGGAGAAGCTTCAAAGACT
GTCATGGAACAAACAAGAACCTTATTTCATGGCTTTATTTGCCGAGAACA
GTCAAGAAGTTCAAATTCTTGACATTCGTATGCCATGCAACATTCTCTGT
CGACTCCGTAATCACACTGGACCAGTGAACGGAATTGCATGGGCCCCACA
CAGTCCACATCACATTTGTACTGCTGGAGACGATTCACAAGCTCTGATTT
GGGATCTGCAGCAGGTTCCAAGACCCGTTGATGATCCGATTCTGGCTTAT
TCGGCTGGTGGAGAGgtgagcatttgaatttgaacttaaaatcttatccg
atcaagatcaaaaagtagtgagattaaaaaatatatatacctgaaaatta
200
400
600
800
1000
aaacgaatgctgaaagagaactttcaaaagttgacactaacgattacaaa
aaaaaaacatttacacgcagttatttgaatttaattataaaaatctcatt
cattcattgaacattttacctgaaaattgaataaaaaggtaaaaaaggtg
Cgtccctttttgttaattttttgcatcaaattttcttatatttcttagaa
ttttacataattttctacaatgtcactagtgggactcagaggtgcagtct
taaaaattgcaagtaatgagccactttgaaaactggaatatttgagatct
gagagtttaaaaactgtttttcgaagcggattgtttttattctaccatgt
Ttacattacatttgctgaaacagttaacaattacatccaagttttttctt
ctatgccgaacttagcttggctccgttttcggggtgaatttttttccacc
tgcttttccggaatcataaacatcgaagtttcatactacgttcagctgtt
gcacgtagtttcttagaaaacataaactatgaattgtcacctaaattttg
ttggagatttccagtttcaaaactttcattactatacaatttccagGTCA
ATCAAATTCATTGGGGTCCTGTACACAGTAACTGGATTGCAATTTGCTTC
AACAAAACTCTTGAGATTCTTCGAGTTTGAGCAGAGGATGTGCGTTCTGT
TTGAGAACTAACAAAAATTTCTCACATTTCTCAATTAACGTTATTTTCCA
TTTCCTGTTATGTTTTTATGTTTTTAAATTATAATTTTTTGTCAATTGCT
CTGATTTCAAGTCACTTTCTCCGCCTTTGTTGTGAACAACTTATTTGAAT
TAATTCCAAAGCATCAGTATGATGATTCACGATCTCTTTCAAACCTCAAT
TTTCTCACGCCAACACACAATTGTTTTATCCACCCGAAAATTATTGTTTT
ATATCCTATTTTTTTCCATTTTCCATTGTGATAAGGTTACAGGAAACCCG
AAAATTTTTTAAGGTGGTTTCAAAAAAAATTTCAGAAATTGT
1200
1400
1600
1800
Figure 2
2000
2200
2400
2600
2800
2842
A
MATNGHAVNGEAHVMQQVAQQPAPPGPLAPQGIGRQNEIVIGGRDRRCEI
YKFTSDQQLYASAWSNKNDIKFRLAVGTVSDVSVNPCAANKVSIVQLKDE
TGELVETASFPMEFPANAVGFIPDPDNVYPDLIATTSDCLRLWRVVDGKV
HPDAVMINNTNSQYGSALTSFDWNELEPRYIGVSSVDTTCTIYDVEVGCA
IGQTKPTAPFTLKTQLIAHDKPVHDIEFAKINGGRDHFATVGADGSARMF
DLRHLNHSTIVYEDPNKEKLQRLSWNKQEPYFMALFAENSQEVQILDIRM
PCNILCRLRNHTGPVNGIAWAPHSPHHICTAGDDSQALIWDLQQVPRPVD
DPILAYSAGGEVNQIHWGPVHSNWIAICFNKTLEILRV*
removed by ok267
Figure 3
WD
CTD
WD
WD
WD
WD
WD
SWAN-1
WD
WD
B
388
100
200
300
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V
V
V
V
V
V
V
V
N
N
N
N
N
N
N
N
N
N
G
G
G
G
G
A
A
A
G
G
I
L
I
I
I
I
I
V
I
I
A
S
A
A
A
A
A
K
K
S
W
W
W
W
W
W
W
W
W
W
A
A
A
A
A
A
A
M
H
A
P
P
P
P
P
P
P
P
P
P
H
H
H
H
H
Q
H
G
T
H
S
S
S
S
S
S
S
S
K
S
P
G
S
S
S
C
S
K
R
S
H
S
C
C
C
R
C
S
N
C
H
H
H
H
H
H
H
K
V
H
I
I
I
I
I
I
I
L
L
I
C
C
C
C
C
C
C
A
L
C
T
T
T
T
T
S
T
T
S
T
A
A
A
A
A
G
A
C
C
V
G
G
G
A
A
G
G
G
G
S
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
S
Y
H
H
H
G
S
C
C
K
A
S
-
S
K
-
P
Q
-
L
V
-
N
C
-
A
K
-
D
T
-
N
L
-
Q
E
-
Y
T
-
S
P
-
P
N
-
L
M
-
L
M
-
S
Y
-
W
A
-
K
N
-
A
A
A
A
A
A
A
L
K
A
G
Q
E
E
E
G
G
E
T
L
G
A
G
G
G
A
A
H
Q
A
E
E
E
E
E
E
E
E
E
E
V
V
V
I
I
I
I
V
I
I
N
N
N
N
N
N
E
N
N
N
Q
Q
Q
N
N
Q
Q
N
N
Q
I
I
I
V
V
L
L
L
I
L
H
H
Q
Q
Q
Q
Q
S
A
S
W
W
W
W
W
W
W
W
W
W
G
S
G
A
A
S
S
S
R
S
P
S
A
S
S
P
S
V
P
S
V
S
T
T
T
A
S
K
Q
S
H
F
Q
Q
Q
Q
Q
N
R
Q
S
P
P
P
P
R
P
G
P
N
D
D
D
D
D
D
D
D
D
W
W
W
W
W
W
W
G
W
W
I
I
I
I
I
I
V
L
F
I
A
S
A
A
A
A
A
A
G
A
I
I
I
I
I
I
I
V
C
I
WD4
WD5
Ce SWAN-1
Ce SWAN-2
Dm SWAN-1
Dr SWAN-1
Hs HAN11
AN11
At AN11
Sp SWAN-1
Sc SWAN-1
Dd SWAN-1
182
272
138
137
137
145
152
183
260
108
G
G
G
G
G
G
G
V
I
A
V
T
T
T
T
T
T
V
S
T
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
V
I
I
I
I
I
T
L
I
I
D
D
D
D
D
D
D
D
D
D
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
I
T
I
V
I
I
I
I
I
V
V
I
Y
W
W
W
W
W
W
W
W
W
D
Q
G
G
G
D
D
D
D
N
V
L
L
L
L
V
I
I
L
I
E
E
E
E
E
E
E
V
Q
E
V
T
T
T
T
K
R
A
S
T
G
G
G
G
G
Q
S
G
Ce SWAN-1
Ce SWAN-2
Dm SWAN-1
Dr SWAN-1
Hs HAN11
AN11
At AN11
Sp SWAN-1
Sc SWAN-1
Dd SWAN-1
265
355
219
219
219
212
219
267
344
176
P
P
P
P
P
P
S
P
P
P
N
Q
A
Q
Q
T
E
P
S
S
P
P
V
S
-
K
-
G
-
K
R
H
H
H
D
D
S
S
F
E
Q
T
H
H
T
T
A
D
V
K
P
A
P
P
P
P
P
A
P
L
L
L
L
L
L
L
L
L
L
Q
L
L
L
L
L
V
L
L
L
R
R
R
R
R
R
R
R
R
R
L
L
L
L
L
L
L
L
L
L
S
A
A
C
C
A
G
S
E
C
W
W
W
W
W
W
W
A
P
W
N
N
N
N
N
N
N
C
S
N
K
R
K
K
K
K
K
D
P
K
Q
N
Q
Q
Q
Q
Q
S
Y
Q
E
D
D
D
D
D
D
D
D
D
P
H
P
P
P
L
P
V
P
P
Y
N
N
N
N
R
R
N
N
N
F
Y
Y
Y
Y
Y
Y
L
V
Y
M
I
L
L
L
M
M
M
L
L
A
A
A
A
A
A
A
A
A
A
L
T
T
T
T
T
T
T
T
T
F
F
V
M
M
I
I
F
F
I
A
G
A
A
A
L
I
H
A
Q
E
Q
M
M
M
M
M
H
A
Q
N
D
D
D
D
D
D
N
D
D
S
S
S
G
G
S
S
S
S
S
Q
K
C
M
M
N
A
S
N
P
E
E
E
E
E
K
K
D
K
K
V
V
V
V
V
V
V
V
I
V
Ce SWAN-1
Ce SWAN-2
Dm SWAN-1
Dr SWAN-1
Hs HAN11
AN11
At AN11
Sp SWAN-1
Sc SWAN-1
Dd SWAN-1
348
438
302
302
302
296
305
351
444
259
P
E
T
-
P
P
A
A
A
N
G
P
S
P
V
I
I
I
I
G
G
S
L
I
D
N
E
E
E
I
L
P
E
E
D
D
D
D
D
D
D
A
D
D
P
P
P
P
P
P
P
P
P
P
I
I
I
I
I
M
I
T
D
L
L
L
L
L
L
S
L
L
G
L
A
A
A
A
A
M
A
S
D
T
Y
Y
Y
Y
Y
Y
Y
V
T
Y
S
R
T
T
T
S
T
S
E
N
A
G
M
-
T
T
-
T
D
-
P
G
-
G
G
-
M
A
-
T
G
-
G
S
-
S
G
-
T
L
-
S
N
-
E
E
-
Y
-
V
-
T
-
P
-
V
-
S
-
S
D
-
V
P
-
N
L
-
WD6
F
F
F
F
F
F
F
F
F
F
A
A
A
A
A
A
A
V
A
A
T
S
S
S
S
S
S
S
S
S
WD7
WD8?
S
S
-
M
L
-
R
N
-
E
N
-
T
N
-
CTD
C
C
C
C
C
A
A
V
V
A
F
S
Y
Y
Y
F
F
Y
S
F
N
D
N
N
N
S
S
G
G
S
K
N
K
N
N
N
T
K
K
S
Figure 3
A
B
wild-type
ced-10(G12V)
C
Figure 4
D
Figure 4
A
B
severe
mild
C
Figure 5
A
Figure 6
B
C
A
Figure 7
Rac1(Q61L)
B
Merge
C
Figure 7
MYR::UNC-115
D
A
B
Figure 8
C
Y190
D
CED-10
E
MIG-2
F
actin
actin
actin
actin
DAPI
DAPI
DAPI
DAPI
G
actin
DAPI
CED-10
Figure 8
RAC-2