1 Department of Molecular Biology, Princeton University, Princeton, NJ
08544-1014, USA
2 Molecular Neurobiology Program, Department of Pharmacology, Skirball
Institute, New York University School of Medicine, 540 First Avenue, SKI 5-17,
New York, NY 10016, USA
* Author for correspondence (e-mail: jschwarzbauer{at}molbio.princeton.edu)
Accepted 10 June 2003
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Summary |
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Key words: C. elegans, Talin, ß integrin, Focal adhesion
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Introduction |
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An overt post-embryonic phenotype caused by inactivation of integrins is
malformation of the gonad arms (Baum and
Garriga, 1997; Lee et al.,
2001
). Severe gonad morphological defects are easily visible with
low-power microscopy, and we used this phenotype as the basis for an RNA
interference (RNAi)-based genetic screen for molecules involved in
integrin-related pathways. RNAi is an effective method for analyzing gene
function in C. elegans as it phenocopies loss-of-function phenotypes
for the gene in question (Fire et al.,
1998
; Timmons et al.,
2001
). In RNAi, double stranded RNA (dsRNA) introduced into larvae
or adults activates an enzymatic pathway that eliminates endogenous RNAs
homologous to the dsRNA (Hannon,
2002
). In this screen, we isolated the gene encoding talin, a
protein that localizes to dense bodies and focal adhesions
(Critchley, 2000
). At these
sites, talin binds to integrin ß cytoplasmic tails, vinculin and actin
filaments (Calderwood et al.,
1999
; Critchley,
2000
). The vital role of talin in embryogenesis is illustrated by
the talin mutant mouse, in which embryos die at day E8.5 from a failure in
embryonic mesoderm formation (Monkley et
al., 2000
). In Drosophila, clones of talin-negative cells
in the wing fail to adhere between layers causing a wing blister and embryos
lacking talin exhibit muscle detachment and failure of germband retraction
(Brown et al., 2002
). In C.
elegans, talin is found in muscle dense bodies along with integrins, and
PAT-3/ß integrin is required for talin localization to these structures
(Moulder et al., 1996
). The
functions of talin in muscle and other nematode tissues have not been
determined.
Gonad morphogenesis is dependent on migration of a specialized gonadal
leader cell, the distal tip cell (DTC)
(Blelloch et al., 1999;
Hubbard and Greenstein, 2000
;
Lehmann, 2001
). Our analysis
shows that loss of talin caused several gonad defects, including inappropriate
migration of the hermaphrodite DTCs, disruption of the actin cytoskeleton in
gonad sheath cells, and oocyte maturation defects in the proximal gonad.
Reduction of talin also resulted in progressive uncoordination and paralysis,
a phenotype that was also observed with reduction of integrin pat-2
and pat-3 RNA levels. Immunofluorescence staining showed perturbation
of muscle filament structure in the paralyzed adults. Thus, talin is an
essential gene that participates in cell migration and contractile functions
in C. elegans larval and adult tissues.
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Materials and Methods |
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In order to generate talin::GFP(zdIs15) nematodes, nucleotide
sequences upstream of the talin gene (-2070 to +6, where +1 is first base of
ATG) were amplified from N2 genomic DNA using PCR and cloned into the
SphI and BamHI sites of the GFP expression vector pPD95.77
(provided by A. Fire) to generate the talin::gfp reporter.
Extrachromosomal arrays were generated by germline transformation of
lin-15(n765) animals with talin::gfp DNA (50 µg/ml) and
lin-15(+) gene (pSK1) (50 µg/ml), and stable chromosomal
integration was induced by treatment with TMP and UV. lin-15(n765)
mutants exhibit a temperature-dependent multivulva phenotype that can be
rescued by pSK1 (Clark et al.,
1994). The talin::gfp(zdIs15) integrant (MT4015) was
backcrossed three times with N2.
Starved nematodes were allowed to recover on fresh E. coli
OP50-seeded NGM plates for two days. This procedure partially synchronizes the
nematode cultures, and guarantees gravid animals for egg collection. Eggs were
released from gravid hermaphrodites using alkaline hypochlorite solution
(Hope, 1999). Following washes
in M9 buffer, eggs were then transferred to plates seeded with RNAi HT115(DE3)
bacteria expressing dsRNA.
RNAi and analysis of phenotypes
The RNAi feeding protocol was essentially as described
(Timmons et al., 2001).
Briefly, bacteria were cultured overnight in LB supplemented with 40 µg/ml
ampicillin, and seeded onto NGM agar supplemented with carbenicillin (25
µg/ml) and IPTG (1 mM). Double-stranded RNA expression was induced
overnight at room temperature on the IPTG plates. Eggs were then transferred
onto the plates and RNAi phenotypes were monitored at varying times.
The talin clone from well 8M01 of the C. elegans Chromosome I
library is a genomic fragment of talin (Gene pairs name: Y71G12A_195.e) cloned
into the RNAi feeding vector pPD129.36
(Fraser et al., 2000) and
transformed into HT115(DE3), an RNase III-deficient E. coli strain
with IPTG-inducible T7 polymerase activity
(Timmons et al., 2001
). The
empty pPD129.36 vector in HT115(DE3) was used as a control.
To construct the pat-2 (F54F2.1) RNAi clone, primers at base positions 5 and 386 relative to the ATG in the predicted cDNA sequence were used for RT-PCR amplification with N2 total RNA as the template. Primers included NheI and HindIII sites at the 5' and 3' ends, respectively, and these sites were used to clone the PCR product into pPD129.36 vector. For the pat-3 (ZK1058.2) RNAi clone, primers at positions 2073 and 2403 in the gene were used for amplification. NcoI and PstI sites subsequently introduced at the 5' and 3' ends were then used to clone the PCR product into pPD129.36. Primers used were:
pat-2 5F 5'-AGGCTAGCGAGAGGGTAGTTTTCCGCG-3'pat-2 386R 5'-AGAAGCTTGATCGAACAGTTGCACC-3'
pat-3 2073F 5'-CTCAACGAAACTACACCCTGCC-3'
pat-3 2403R 5'-TTAGTTGGCTTTTCCAGCGTATACTGG-3'
To characterize phenotypes, eggs were hatched on bacteria expressing dsRNA and larvae and adults were examined using a dissecting microscope. Observed phenotypes included Unc (uncoordinated) movement, paralysis, large clear patches in the body cavity, Egl (egg-laying defective) and sterile.
To analyze gonad morphology, young adult or L4 hermaphrodites were mounted in a drop of M9 buffer containing 0.1 M sodium azide on a cover slip coated with 2% agarose and examined using a Nikon Eclipse TE 2000-U microscope with DIC optics. Defects such as inappropriate or extra turns, migration in the wrong direction, or aberrant stops were counted as DTC migration anomalies. Oocyte accumulation was scored when oocytes were present in a non-linear arrangement in the proximal gonad. Paralysis was scored when animals were able only to move their heads.
Standard errors for the proportions of defective DTC migration, oocyte
stacking and paralysis in a population were calculated using the observed
frequency and sample size, assuming a binomial distribution as previously
described (Hedgecock et al.,
1990).
Fluorescence microscopy
Fluorescence microscopy was performed with a Nikon Eclipse TE 2000-U
microscope equipped for epifluorescence. Images were captured with a Cooke
SensiCam High Performance camera using IP lab software (Scanalytics).
DNA organization in the gonads was monitored in animals stained with DAPI
(0.01 µg/ml). To visualize muscle structure, RNAi-treated and control whole
nematodes were frozen on poly-L-lysine-coated slides and fixed with methanol
and acetone as described (Lee et al.,
2001). Fixed animals were stained at room temperature with MH25
anti-PAT-3 antibodies (1:250 dilution) for 4 hours at room temperature,
followed by rhodamine-conjugated goat anti-mouse IgG (Molecular Probes) for 2
hours at room temperature. Actin cytoskeletal organization of muscle cells was
examined by staining fixed animals with 0.4 U/ml of rhodamine-conjugated
phalloidin (Molecular Probes) for 2 hours at room temperature.
Nematode gonads were dissected, fixed and stained as described
(McCarter et al., 1997;
Strome, 1986
). Briefly,
animals in PBS containing 0.2 mM levamisole were decapitated with syringe
needles resulting in gonad extrusion. For visualization of actin, dissected
gonads were fixed in formaldehyde for 2 hours, extracted with ice-cold acetone
for 3 minutes, washed in PBS and incubated in 2 U/ml rhodamine-phalloidin
(Molecular Probes) for 1 hour. Dissected gonads were then transferred to
slides coated with 2% agarose and visualized as described above.
Real time RT-PCR
RNA from mixed-stage populations of nematodes was isolated. Briefly,
animals were collected in M9 buffer, and pelleted through a 4% sucrose
gradient to remove bacteria. After grinding with 3.5 g glass beads (Sigma) in
2.5 ml of Trizol (Invitrogen), the pellet was extracted twice in Trizol and
1/5 volumes of chloroform. RNA was precipitated with isopropanol, rinsed with
ethanol and treated with RQ1 DNase (Promega). RNA (1 µg total) was used to
synthesize cDNA using Superscript reverse transcriptase (Invitrogen) primed
with random hexamers in a 20 µL reaction. For PCR amplification, 1 µL of
cDNA reaction product was used in each reaction with iQ SYBRgreen Supermix
(BioRad) in a 96-well plate. Primer concentrations were optimized according to
the manufacturer's recommendations. Amplification of C. elegans gene
T09F3.3 (GAPDH) was used as a loading and amplification control. All assays
were amplified and evaluated in real time using the iCycler iQ detection
system (BioRad), and the relative quantitation of talin, pat-2 and
pat-3 mRNA expression was calculated by the comparative Ct method
(Livak and Schmittgen, 2001).
Primers used for amplification are listed below. Numbers refer to base
positions within the cDNA relative to the ATG.
Talin 2714F 5'-AGCAAGCAGAATCACAGC-3'Talin 2908R 5'-CGATCTGCTTTCAATGACATC-3'
pat-2149F 5'-ACCATGAGCAGAAGGGAATG-3'
pat-2 240R 5'-ACGGAGCAAGCATAAACTGC-3'
pat-3 347F 5'-CTGAGGAAGAAGCCGTTCAG-3'
pat-3 429R 5'-AATCAACGGCTTGACGGTAG-3'
GAPDH 827F 5'-TGAAGGGAATTCTCGCTTACACC-3'
GAPDH 980R 5'-GAGTATCCGAACTCGTTATCGTAC-3'
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Results |
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During normal C. elegans development, migration of specialized
gonadal leader cells, the DTCs, forms the two arms of the U-shaped
hermaphrodite gonad. Defects in interaction of the DTC with extracellular
substrates and pathfinding cues result in malformed gonad arms
(Hubbard and Greenstein,
2000). Comparison of gonads from N2 nematodes with talin RNAi
animals demonstrated that loss of talin expression induced significant defects
in gonad morphology. Misdirected gonad migration was observed in 61%
(±3%, n=236) of talin RNAi hermaphrodite gonad arms after
48-50 hours of exposure to RNAi bacteria. The most typical defects were
failure to turn and migrate along the dorsal side of the animal. Other defects
included inappropriate turns and premature stops
(Fig. 1C,D). Gonad elongation
along the ventral body wall appeared normal. Defects were rarely observed in
animals fed bacteria containing an empty RNAi vector (6%±2%,
n=154).
Disruption of talin function within the DTC could generate these migration and gonad morphological defects. In order to determine whether talin is expressed in the DTC, transgenic nematode lines that express a talin::gfp transcriptional fusion were prepared. Fluorescence microscopy of transgenic animals showed that GFP was expressed in body wall muscles, sex muscles, DTCs and sheath cells of the somatic gonad (Fig. 2). These results show that talin is appropriately distributed to affect DTC migration.
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Oocyte maturation is defective in the proximal gonad
In wild-type nematodes, oocytes line up at the spermatheca, mature, and are
ovulated in an assembly line fashion. Fertilization occurs as oocytes are
moved through this organ in response to complex signals from the spermatheca,
the oocyte and the sheath cells that surround the gonad
(Schedl, 1997). Stacking of
oocytes occurs when oocytes are unable to move through the spermatheca because
of a failure in proper signaling, or to defective spermatheca or sheath cell
expansion and contraction (Hubbard and
Greenstein, 2000
; McCarter et
al., 1997
; McCarter et al.,
1999
).
We found that oocytes commonly accumulated in the proximal gonad arms of animals with reduced talin levels (Fig. 3B). Most of these animals had at least one gonad arm affected (85%±3%, n=114). This arrangement is in contrast to wild-type oocytes, which are organized in a linear array (Fig. 3A). Germ cell DNA, visualized by DAPI staining, appeared normal in the distal gonad arm and sperm were visible proximal to the oocytes (Fig. 3C). In contrast to control hermaphrodites, germ line nuclei were frequently observed throughout the gonad arms of talin RNAi animals (55%±7%, n=49) (Fig. 3D), perhaps because of a failure to mature into oocytes. By 72 hours of exposure to talin RNAi, the gonads usually ruptured, leading to severely compromised gonad function and sterility of the talin RNAi hermaphrodites.
|
Because talin is expressed in the sheath cells (see Fig. 2), it seemed probable that defective sheath cell cytoskeletal connections to the plasma membrane led to gonad distension by oocyte stacking. In order to observe the actin cytoskeleton in sheath cells, dissected gonads were stained with rhodaminephalloidin. Wild-type gonads showed the typical actin filament pattern demarcating the edges of oocytes as well as extending length-wise throughout the gonad sheath cells (Fig. 4A). Talin RNAi animals had abundant actin filaments surrounding the germ cells but very little organized actin in the proximal gonad (Fig. 4B). Thus, loss of talin leads to a severe reduction of actin cytoskeletal organization in the proximal gonad.
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Loss of talin leads to total body muscle paralysis
In addition to the gonad-related phenotypes, talin RNAi resulted in
progressive and ultimately complete paralysis. By 58 hours post-hatch, the
majority of the nematodes became paralyzed
(Fig. 5A), able only to move
their heads (Fig. 5B). By 72
hours, almost 100% of the animals were paralyzed. C. elegans body
wall muscles consist of rows of cells that extend along the body and form
multiple attachments to the basement membrane
(Moerman and Fire, 1997).
Talin is localized to muscle dense body complexes that form a regularly
repeated dotted pattern along the length of muscle cells
(Moulder et al., 1996
).
Indirect immunofluorescence staining of the integrin ß subunit PAT-3 was
used to visualize dense body organization. By 72 hours of talin RNAi, PAT-3
organization appeared very irregular and disordered and the muscle structure
was significantly disrupted (Fig.
5C,D). Contraction of muscles with weakened cytoskeletal
attachments probably results in the observed irregular patches in the muscle
cells.
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Talin functions with PAT-2 integrins in muscle
C. elegans has two integrin subunits, INA-1 and PAT-2,
which primarily function in distinct tissues. INA-1 is expressed in many
non-muscle cell types including DTCs, uterine, vulval and neuronal cells
(Baum and Garriga, 1997
).
Several viable ina-1 mutant alleles have similar misdirected or
misshapen gonad arms and mislocalized germ cells as seen in talin-depleted
animals, suggesting that talin works in concert with INA-1/PAT-3
ß
heterodimers in these tissues. Although the tissue distribution and mutant
phenotypes of pat-2 have not been published, pat-2 has been
shown to be essential for muscle development
(Williams and Waterston,
1994
). Therefore, we predicted that depletion of PAT-2 would
result in muscle phenotypes similar to those of talin-depleted animals.
Reduction of either PAT-2 or PAT-3 by RNAi caused muscle-related phenotypes
identical to talin RNAi. Within 72 hours, paralysis was accompanied by severe
disorganization of body muscle actin filaments
(Fig. 6). Reduction of PAT-2
levels also led to an egg-laying defect, with animals developing the
bag-of-worms phenotype by 72 hours (75%±4% n=95). Unlike talin
loss-of-function, however, gonad morphology was not visibly perturbed by
pat-2 RNAi. Real time reverse transcriptase polymerase chain reaction
(RT-PCR) analysis confirmed that defective animals had significantly reduced
levels of talin, pat-2 and pat-3 RNA by 48 hours
(Fig. 7). Talin RNA levels were
reduced by 48 hours and remained low throughout the experimental time course.
In addition, we verified that levels of pat-2 and pat-3 RNA
do not change in response to talin RNAi at 48 hours (data not shown). These
results indicate that PAT-2/PAT-3 integrins are working in concert with talin
in C. elegans muscle whereas INA-1/PAT-3 integrins function with
talin during DTC migration.
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Discussion |
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In talin RNAi animals, we commonly observed DTCs that had failed to turn
and migrate toward the midpoint on the dorsal ECM. This phenotype is similar
to the abnormal gonad morphology we observed with dominant negative inhibition
of integrins in the DTC (Lee et al.,
2001). Talin binding to the ß integrin cytoplasmic tail
allows integrin activation and ligand binding in cell culture systems, perhaps
by relieving an inhibitory interaction between the
and ß integrin
subunits (Calderwood et al.,
2002
; Calderwood et al.,
1999
). Thus, talin reduction by RNAi will probably affect the
activity of integrins. Failure of the DTCs to complete the appropriate
migratory path could be due to ineffective cell adhesion, or to incorrect
detection or interpretation of directional cues positioned within the dorsal
ECM. Talin may also be required for initial DTC migration along the ventral
ECM, but this might not be evident by talin RNAi because larvae may contain
residual talin protein synthesized during embryogenesis. Proper formation of
hermaphrodite gonad arms depends on directional cues from proteins such as
UNC-6/netrin and UNC-40/netrin receptor for ventral to dorsal migration
(Ishii et al., 1992
), and
UNC-129/TGF-ß on the dorsal side
(Colavita et al., 1998
).
Coordination between these signals and integrin-talin connections to the
cytoskeleton appear to control directed movement during gonad
morphogenesis.
Our results support the hypothesis that the integrin ß
PAT-2/PAT-3 heterodimer is the functional integrin required in nematode
muscles. Furthermore, we have shown that this integrin pair acts through talin
to organize and maintain the muscle actinomyosin contractile structure.
Depletion of talin, PAT-2 or PAT-3 resulted in paralysis and breakdown of the
muscle actin structure by 72 hours. Embryos homozygous for loss of function
mutations in various genes of the C. elegans dense body including
deb-1/vinculin, pat-2/
-integrin,
pat-3/ß-integrin, pat-4/ILK and
pat-6/
-parvin exhibit similar Pat phenotypes and embryonic
lethality (Williams and Waterston,
1994
). In these animals, muscle cell actin and myosin are not
organized into sarcomeres, and dense body and M-line components fail to
assemble. Animals depleted of talin by RNAi show similar muscle phenotypes,
suggesting the null phenotype of talin will probably be Pat.
Oocyte maturation, ovulation and fertilization must occur in the proper
sequence and in coordination with the somatic gonad in order to produce viable
progeny. Ablation of sheath cells results in an impairment of germ cell
meiotic progression and gametogenesis
(Schedl, 1997), a process that
involves MAP kinase signaling (Church et
al., 1995
; Miller et al.,
2003
). Ovulation is dependent on sheath cell contractions that
move mature oocytes through the spermatheca
(Schedl, 1997
). We found that
the sheath cell actin cytoskeleton of talin RNAi animals was severely
disorganized. Compromised sheath cell function blocked germ cell maturation
and allowed gonad distension and oocyte accumulation in the proximal gonad
arms. These results demonstrate that talin plays essential roles in multiple
sheath cell functions during oocyte development and ovulation.
Talin loss of function phenotypes show considerable similarities to
previously reported pat-3 integrin defects
(Gettner et al., 1995;
Lee et al., 2001
;
Williams and Waterston, 1994
).
In addition to talin, integrins interact with many intracellular proteins that
play key roles in integrin function (Liu
et al., 2000
), and we suspected loss of some of these proteins
might result in phenotypes similar to those of talin RNAi. The C.
elegans genomic database of RNAi results was searched for those genes
giving a paralyzed (Prz) RNAi phenotype
(Kamath et al., 2003
). Of the
49 genes that had a Prz phenotype, known cell architecture and cell signaling
genes were over-represented with 38% compared with 21% of all genes with RNAi
phenotypes. Among these, loss of pat-4/ILK resulted in cell
contraction phenotypes that most closely resembled talin RNAi, including
uncoordination, paralysis and lethality. Whether ILK and other Prz genes
participate in cell migration in addition to their roles in contractile cell
functions remains to be determined.
Our results show that talin plays important roles in post-embryonic
organogenesis and tissue functions. Furthermore, talin functions with the
ß PAT-2/PAT-3 heterodimer, and probably the
ß
INA-1/PAT-3 heterodimer, for critical tissue- and stage-specific functions. In
addition, this work demonstrates that screening for defects in DTC migration
using bacterial RNAi can rapidly identify gene products that affect integrin
signaling and function.
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Acknowledgments |
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