1 Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston,
MA 02115, USA
2 Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115,
USA
* Author for correspondence (e-mail: perrimon{at}rascal.med.harvard.edu)
Accepted 21 November 2002
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SUMMARY |
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Key words: Rac, Rho, Rho kinase, Myosin II, Myosin phosphatase, zipper, spaghetti-squash, MYPT, Dorsal closure, Ring canal growth, Eye morphogenesis, Drosophila melanogaster
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INTRODUCTION |
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The activity of smooth muscle/non-muscle myosin II is regulated by the
phosphorylation of MRLC that is modulated by the antagonistic activity of
myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP).
MLCP is composed of three subunits: a catalytic subunit made up of protein
phosphatase 1c ß (also called ), a myosin binding or targeting
subunit (MYPT), and a small subunit of unknown function. MYPT binds and
confers the selectivity of PP1c for myosin
(Hartshorne et al., 1998
).
The phosphatase activity of MLCP can be regulated in several ways (reviewed
by Hartshorne et al., 1998;
Somlyo and Somlyo, 2000
).
Rho-kinase (ROCK) phosphorylates an inhibitory phosphorylation site on MYPT
and inhibits the phosphatase activity in smooth muscle. This phosphorylation
may occur through ZIPK (leucine zipper interacting protein kinase)-like kinase
(MacDonald et al., 2001
) or
integrin-linked kinase (Kiss et al.,
2002
). Myotonic dystrophy protein kinase phosphorylates the same
inhibitory phosphorylation site (Muranyi
et al., 2001
), although it is not clear whether this
phosphorylation event also goes through ZIPK. In addition, protein kinase C
(PKC) can phosphorylate the ankyrin repeat region of MYPT, and thus attenuate
the interaction of MYPT with PP1c and MRLC
(Toth et al., 2000
).
Furthermore, CPI-17, a smooth muscle-specific inhibitor of MLCP, can also
regulate the phosphatase activity of MLCP. Phosphorylation of CPI-17 by PKC,
or ROCK, or protein kinase N, or p21-activated kinase (PAK) dramatically
enhances the inhibition ability of CPI-17
(Eto et al., 1997
;
Koyama et al., 2000
;
Senba et al., 1999
;
Takizawa et al., 2002a
;
Takizawa et al., 2002b
).
Finally, MRLC can also be phosphorylated by ROCK and PAK, which itself is a
substrate of Rac and Cdc42. Thus ROCK can regulate MRLC phosphorylation both
through direct phosphorylation of MRLC and through inactivation of MLCP.
Importantly, although the biochemistry of these phosphorylation events is well
characterized, the physiological significance of these regulatory steps in
vivo remains to be explored.
The in vivo function of non-muscle myosin II has been extensively analyzed
in Drosophila melanogaster, Dictyostelium discoideum and
Saccharomyces cerevisiae. Drosophila has a single non-muscle myosin
II heavy chain encoded by zipper (zip), as well as a single
non-muscle myosin II regulatory light chain encoded by spaghetti squash
(sqh). Analysis of the phenotypes associated with mutations in
zip and sqh have revealed that non-muscle myosin II
regulates cell shape changes and cell movements in multiple processes such as
cytokinesis, dorsal closure and oogenesis
(Edwards and Kiehart, 1996;
Jordan and Karess, 1997
;
Wheatley et al., 1995
;
Young et al., 1993
). In
addition, mutations in both zip and sqh affect planar cell
polarity during development (Winter et
al., 2001
).
The temporal requirement of zip has been studied in
sqh2 mutant animals that carry a sqh transgene
driven by a heat shock promoter (Edwards
and Kiehart, 1996). This analysis showed that sqh
activity is needed for eye and leg imaginal discs morphogenesis. Also, during
oogenesis, sqh is required for morphogenesis of interfollicular
stalks, border cell migration, centripetal cell ingression, dorsal appendage
cell migration, and rapid transport of the nurse cell cytoplasm into the
oocyte. Inhibition of this transport was also observed in animals that carry
homozygous sqh1 germline clones (GLCs)
(Wheatley et al., 1995
).
The in vivo function of MRLC phosphorylation was determined by expression
of sqh transgenes that contain mutated phosphorylation sites in a
sqh null mutant background
(Jordan and Karess, 1997).
Embryos carrying the null mutation sqhAX3 die, mostly
during the first larval instar, and sqhAX3 GLCs develop
extensive defects, including failure in cytokinesis, during oogenesis.
SqhA20A21, which has both the primary and secondary phosphorylation sites
changed to alanine, failed to rescue sqhAX3, indicating
that phosphorylation of Sqh is important for myosin II function. In support of
this, a change of serine 21 to glutamic acid (SqhE21), that presumably mimics
constitutive phosphorylation of Sqh, substantially rescues the
sqhAX3 oogenesis phenotype.
To gain further insight into the regulation of Zip and to define precisely the in vivo function of MLCP, we have cloned the Drosophila homologue of the MYPT gene (DMYPT). We find that DMYPT is essential for cell sheet movement during dorsal closure, morphogenesis during eye development, and ring canal growth during oogenesis. Our results indicate that regulation of the phosphorylation state of MRLC, and dynamic activation and inactivation of myosin II, are essential for its various functions during many developmental processes.
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MATERIALS AND METHODS |
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Cloning of DMYPT
The DMYPT cDNAs, AT12677, RE34228, RE63915 and AT31926 were
obtained from ResGen, Invitrogen Corporation. AT12677 was sequenced completely
in both directions, while the other clones were only sequenced from their most
5' and 3' ends. To determine the organization of the
DMYPT locus, sequences from all clones were assembled into one contig
and then compared with the Drosophila genome sequence. AT12677,
RE34228, RE63915 contain the entire DMYPT open reading frame (ORF).
RE34228, RE63915 include all of exon 1, AT12677 starts from the middle of exon
2, 43 bp 5' of the start codon. AT31926 starts within exon 4. AT12677,
RE34228 and AT31926 share the same 3' end.
Generation and rescue of DMYPT mutations
P-element insertions were excised using the 2-3 transposase
following conventional methods. These excision lines were then analyzed for
lethality and fertility. For rescue experiments, the DMYPT ORF from
AT12677 was cloned by PCR into CaSpeR-hs between NotI and
XbaI, and injected with helper DNA (a source of
2-3
transposase) into w1118 flies to generate transgenic
lines. One of the transgenic lines, hs2-4, is a viable insertion on
the X chromosome and was used to generate hs2-4/+;
DMYPT03802/TM3, Sb animals. The numbers of rescued
DMYPT03802 mutant progeny were scored following a 1-hour
daily heat-shock treatment at 37°C.
Generation of germline clones
Germline clones (GLCs) of DMYPT03802 were generated as
described previously (Chou and Perrimon,
1996), by crossing y w hs-FLP22;
ovoD1FRT2A/TM3, Sb males to
DMYPT03802FRT2A/TM3, Sb females. Third instar
larval progeny were placed at 37°C for 2 hours each day for 3 days.
Females with DMYPT03802 homozygous GLCs were mated with
DMYPT03802/TM3, actinGFP males and allowed to lay
eggs.
Cuticle and eggshell preparation
For cuticle preps, overnight egg collections were aged for 30 hours at
25°C, dechorionated with a 50% solution of commercial bleach, washed with
PBST (PBS and 0.1% Triton X-100), mounted in Hoyers mounting medium with
lactic acid, and heat-treated at 60°C overnight. Eggshells were prepared
similarly without the bleaching step. Images were taken with a SPOTTM
digital camera (Diagnostic Instruments) using phase-contrast or dark-field
optics on a Zeiss Axiophot microscope and processed with Adobe Photoshop.
Dissecting and staining of egg chambers
Egg chambers were dissected in Schneider's Drosophila medium with
10% FBS, fixed (PBS with 0.5% Triton X-100, 6 U/ml of Texas Red phalloidin, 4%
formaldehyde) for 20 minutes, washed with PBST, incubated with primary
antibodies in PBS with 0.3% Triton-X-100 and 0.1 µg/µl of BSA, at
4°C overnight, washed, and incubated with secondary antibodies at room
temperature for 1.5 hours. Texas Red phalloidin (2 U/ml) and DAPI (1 µg/ml)
was added during the last half an hour incubation, and washed with PBST.
Primary antibodies used were: anti-Kelch 1B (1:1)
(Xue and Cooley, 1993),
anti-hu-li tai shao RC (anti-Hts) (1:1)
(Robinson et al., 1994
) (both
from Developmental Studies Hybridoma Bank), anti-Zip
(Jordan and Karess, 1997
), and
anti-phosphotyrosine (4G10, 1:500, UBI). Secondary antibodies used were: Texas
Red, Cy5, or Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG (Jackson or
Molecular Probes). Egg chambers were staged according to Spradling
(Spradling, 1993
). Images were
captured with a Leica TCS-NT confocal microscope and a series of Z sections
were stacked.
Embryo staining
In situ hybridization to embryos was performed as described previously
(Patel et al., 1987). For
immunofluorescence staining, embryos from mutant/TM3,actinGFP were
fixed and stained with rabbit anti-GFP serum (1:2000, Molecular Probes) and
mouse anti-fasciclin III (7G10, 1:20, DSHB) or anti-phosphotyrosine (4G10,
1:1500, UBI). Homozygous mutant embryos were identified by the absence of
GFP.
Genetic interaction
Males carrying GMR-Rac7A were crossed to females of the
following genotypes: OreR, Drok2/FM7, zip1/Cyo,
ckP13/Cyo, DMYPT2-188/TM3, Sb Tb,
DMYPT2-199/TM3, Sb Tb, DMYPT03802/TM3, Sb Tb,
Df(3L)th102/TM3, Sb, y w;sqh[E20E21], w;sqh[A20A21], and
RhoA720/CyO. The progeny were raised at 29°C and the
resulting adults were dehydrated in an ethanol series, dried in SAMDRI PVT-3B
and coated with Hummer V Sputter Coater. Scanning electron micrographs were
generated using a LEO 1450 VP electron microscope.
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RESULTS |
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Genetics of the DMYPT locus
To characterize the consequences of loss of DMYPT function during
development, we searched for mutations in the DMYPT gene. Two
P-element transposon insertions in the DMYPT locus have been defined
molecularly by recovery of flanking genomic sequence
(Fig. 1A). EP(3)3727,
in the first intron, is homozygous viable and l(3)03802, in the tenth
intron, is associated with zygotic lethality. We also identified several
deficiencies that remove DMYPT sequences based on genetically defined
breakpoints as well as their failure to complement l(3)03802
(Fig. 1D). Df(3L)th102
deletes DMYPT entirely and thus serves as a complete loss-of-function
allele for use in this study.
To determine whether the l(3)03802 P-element insertion within the
DMYPT locus is responsible for the lethality, and to generate new
deletion alleles, we excised both DMYPT P-element insertions using
the 2-3 transposase. Mobilization of each element resulted in the
recovery of both viable precise excisions and lethal imprecise excisions.
Among the >200 excisions derived from l(3)03802, over half were
viable, indicating that the lethality associated with the l(3)03802
chromosome is due to disruption of DMYPT and not another lethal hit.
Thus l(3)03802 is renamed as DMYPT03802 and
EP(3)3727 as DMYPT3727. Two of the strongest
embryonic lethal excision lines, DMYPT2-188 and
DMYPT2-199, like the original insert,
DMYPT03802, fail to complement Df(3L)th102 and
are described in detail below. Eleven of the 39 lethal excisions derived from
DMYPT3727 failed to complement with
DMYPT03802 and Df(3L)th102, which is consistent
with the notion that they disrupt DMYPT activity.
To confirm that the DMYPT03802 insertion disrupts DMYPT
function and that the cDNA derived from the DMYPT locus encodes all
the functions associated with DMYPT activity, we rescued the original lethal P
insertion with a transgene containing a heat shock promoter driving a
DMYPT cDNA. Following 1-hour heat treatments daily from embryogenesis
to eclosion, hs-DMYPT fully rescues DMYPT03802
homozygous animals to adulthood. Stopping heat treatment 1 to 2 days before
eclosion lead to incomplete rescue of DMYPT03802, with
adults developing wing and leg defects similar to those noted for zip
or sqh mutants partially rescued by a transgene
(Edwards and Kiehart, 1996;
Halsell et al., 2000
) (data not
shown). Stopping heat treatment 3 days prior to eclosion resulted in no rescue
to adulthood. The complete rescue of the lethality associated with
DMYPT03802 by the hs-DMYPT transgene demonstrates
that loss of DMYPT activity is responsible for the lethal
phenotype.
Loss of DMYPT activity during embryogenesis is associated
with a dorsal closure phenotype
To assess the timing and cause of lethality associated with the
DMYPT03802 insertion, embryos were collected and analyzed.
Lethal phase analysis showed that 44% of homozygous
DMYPT03802 animals die during embryogenesis, while the
remaining 56% die during early first larval instar (485 total embryos
counted). More than 80% of the dead mutant embryos displayed a failure of
dorsal closure with a characteristic dorsal hole in their cuticles
(Fig. 2B,C). The size of the
hole in such flies is variable and is also influenced by the genetic
background (data not shown). Homozygous Df(3L)th102 embryos
(Fig. 2D), as well as
DMYPT03802/Df(3L)th102 embryos
(Fig. 2E) also showed dorsal
closure defects. The embryonic cuticle phenotype of
DMYPT03802/Df(3L)th102 is more severe (more embryos
displayed large dorsal holes) than homozygous DMYPT03802,
suggesting that DMYPT03802 is a hypomorphic allele. In
addition, all of the embryonic lethal excision lines analyzed that were
derived from DMYPT03802 (data not shown), and ten of the
lethal excision lines from DMYPT3727
(Fig. 2F), produced embryos
with dorsal closure defects. Altogether, these results indicate that
DMYPT is required for dorsal closure.
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Dorsal closure involves a cell sheet movement where the dorsal-lateral
ectoderm on both sides of the developing embryo moves toward the dorsal
midline to cover a degenerative squamous epithelium, the amnioserosa (reviewed
by Knust, 1997;
Noselli and Agnes, 1999
;
Stronach and Perrimon, 1999
).
This epithelial cell sheet movement encloses the embryo in a continuous
protective epidermis. Genetic loss-of-function studies have identified the Jun
N-terminal kinase (JNK) signal transduction cascade as one of the key
modulators of dorsal closure morphogenesis
(Noselli and Agnes, 1999
).
Transcriptional targets of JNK signaling include decapentaplegic
(dpp), a secreted morphogen related to the bone morphogenetic
proteins (BMPs), and puckered (puc), a dual-specificity
phosphatase that mediates a negative feedback loop of the JNK signal
transduction pathway via dephosphorylation of JNK.
To determine whether the failure of dorsal closure in DMYPT mutants is due to an influence on JNK signaling, we assayed for dpp expression in the leading cells of the ectoderm during closure. In situ hybridization revealed that the spatial and temporal expression pattern of dpp is normal in DMYPT mutant embryos (data not shown), suggesting that DMYPT does not function through the JNK pathway during dorsal closure.
To further examine the cause of dorsal closure defects in the mutants, we stained DMYPT mutant embryos for markers that allowed us to analyze the cell polarity and shape in the dorsal ectoderm. We observed apically localized phosphotyrosine immunoreactivity similar to wild-type flies (Fig. 3A). Moreover, there was normal basolateral fasciclin III immunostaining (Fig. 3B). Altogether, these results suggest that there are no gross defects in cell orientation or polarity. However, we did notice that older mutant embryos began to show abnormal cell shapes at the leading edge of the epidermis (Fig. 3B), which could account for the defects in dorsal closure observed in the DMYPT mutants.
|
Consistent with the late embryonic defects observed in DMYPT zygotic mutants, we find that DMYPT is maternally contributed and ubiquitously expressed during embryogenesis (data not shown). This maternal supply of DMYPT is likely the reason that the dorsal closure phenotype is variable among embryos and is influenced by genetic background. However, we cannot address this question directly since DMYPT is required during oogenesis (see below).
DMYPT is required for ring canal growth during
oogenesis
During oogenesis, each cystoblast divides four times with incomplete
cytokinesis and produces one oocyte and fifteen support nurse cells that are
all connected through cleavage furrows. These cleavage furrows subsequently
develop into ring canals. These open rings allow the nurse cells to transport
cytoplasm into the oocyte, slowly from stage 6 to stage 10, then rapidly at
stage 11. This fast phase of transport is referred to as `dumping', and has
been shown previously to require the activity of Sqh (MRLC). In sqh
mutant germline egg chambers, dumping is blocked
(Wheatley et al., 1995).
To analyze the role of DMYPT during oogenesis, we generated
homozygous mutant germline clones (GLCs) of DMYPT03802
using the FLP-FRT/dominant female sterile technique
(Chou and Perrimon, 1996).
Females carrying DMYPT03802 homozygous GLCs lay few tiny
eggs, about a quarter of the size of wild type eggs
(Fig. 4, compare A and C), which do not develop. Examination of the mutant egg chambers revealed that the
dumping of nurse cell cytoplasm to the oocyte was blocked
(Fig. 4, compare B and D). This
is similar to the dumpless phenotype observed with sqh homozygous
mutant GLCs as well as for mutants in other actin binding proteins (reviewed
by Robinson and Cooley,
1997
).
|
To investigate the basis of the dumpless phenotype associated with DMYPT03802 GLCs, we stained actin filaments using Texas Red phalloidin. The most obvious defect involves the ring canals. At stage 8, wild-type egg chambers had large bagel-shaped ring canals (Fig. 5A). In contrast, the ring canals of DMYPT03802 GLC egg chambers were very small (Fig. 5B).
|
To determine whether the ring canals of DMYPT03802 GLCs
never enlarged, or whether they grew and then collapsed, we examined the ring
canals in different stage egg chambers. In wild-type egg chambers, ring canals
grow from 1 µm at stage 2 to 10 µm at stage 11
(Fig. 5C) (see also,
Tilney et al., 1996). In
contrast, the ring canals of DMYPT03802 GLCs barely grew
(Fig. 5D). Mutation of
DMYPT in follicular cells have no effects on the ring canal growth
(data not shown), suggesting that DMYPT is required in the germline for ring
canal growth. Presumably, these small ring canals cannot support the fast
phase cytoplasmic transport and thus cause the dumpless phenotype resulting in
tiny eggs.
In addition to actin, several other proteins, including Hu-li tai shao
(Hts), Kelch, and phosphotyrosine (pY)-containing proteins
(Robinson et al., 1994;
Xue and Cooley, 1993
), are
recruited to ring canals as they form. Immunolocalization experiments revealed
that both Hts and Kelch were localized to the small DMYPT mutant ring
canals (Fig. 6A,B).
Interestingly, although pY staining was present in the mutant ring canals, we
also observed an ectopic accumulation of pY staining in the nurse cells
(Fig. 6D arrows). The basis of
this ectopic accumulation remains to be determined.
|
Next, we analyzed the subcellular distribution of Zip. It has been reported
that mutation of Sqh caused Zip to form aggregates
(Edwards and Kiehart, 1996;
Jordan and Karess, 1997
;
Wheatley et al., 1995
), thus
we expected to detect an effect on Zip distribution in the absence of DMYPT.
Surprisingly, no major changes in Zip distribution were detectable between
wild-type egg chambers and DMYPT GLCs. In both cases, Zip was
uniformly distributed at low level with enhanced cell cortex localization
(Fig. 6C). Our observations are
consistent with the result that DMYPT mutations have no effect on Zip
localization during dorsal closure (Mizuno
et al., 2002
).
Interaction between DMYPT and the small GTPases during eye
development
Previous studies have shown that the Rho family GTPases, Rac1, RhoA, and
Cdc42, each play a role in dorsal closure
(Glise and Noselli, 1997;
Harden et al., 1995
;
Harden et al., 1999
;
Hou et al., 1997
;
Magie et al., 1999
;
Strutt et al., 1997
), and may
influence myosin activity through a RhoA mediated signal. Programmed
overexpression of these genes by the eye-specific GMR promoter causes distinct
rough eye phenotypes (Hariharan et al.,
1995
; Nolan et al.,
1998
). To pinpoint the relationship of DMYPT with these
GTPases, we examined the effects of reducing DMYPT activity on the
rough eye phenotypes. Interestingly, reduction of DMYPT strongly
enhanced the eye phenotype caused by GMR-Rac7A
(Fig. 7 compare B and C). The
eyes of GMR-Rac7A/DMYPT03802 flies were much
smaller, with fewer bristles and hexagonal-shaped ommatidia, than those of
GMR-Rac7A/OreR flies. Consistent with the idea that the
P-insertion and the excisions are hypomorphic alleles, Df(3L)th102
enhanced the GMR-Rac7A eye phenotype to an even greater
extent than either DMYPT03802, DMYPT2-188 or
DMYPT2-199 (data not shown). However, reduction of
DMYPT had no effect on the size of the rough eye caused by either
GMR-RhoA or GMR-Cdc42, although it did enhance the rough eye
phenotype caused by GMR-RhoA as fewer bristles formed
(Fig. 8 compare B and C).
Together, these data suggest that DMYPT plays a role in eye
development and functions downstream of, or in parallel with Rac and
Rho.
|
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DMYPT is a negative regulator of the Rho/myosin signaling
pathway in vivo
RhoA functions downstream of Rac in determining ommatidia polarity in the
eyes (Fanto et al., 2000).
Reducing the dosage of RhoA enhances the effect of
sev-RacN17, a dominant negative form of Rac driven by the
sevenless (sev) enhancer-promoter in the eye, and suppresses
the activity of sev-RacV12, which encodes a constitutively
active form of Rac. Consistently, overexpression of RhoA
(sev-RhoA) rescues sev-RacN17, while reduction
the amount of Rac using a deficiency that uncovers Rac has
no effect on the gain-of-function RhoA phenotype. Thus, similar to
the Rho dependence on Rac function observed in mammalian fibroblasts, some
developmental events in Drosophila also rely on a hierarchy of GTPase
function (Nobes and Hall,
1995
).
Consistent with these observations, reducing the dosage of RhoA partially suppresses the rough eye phenotype caused by GMR-Rac (Fig. 7, compare B and D). In fact, mutations of all the putative positive regulators of myosin activity (RhoA-Zip signaling pathway), including RhoA, Drok and zip itself, moderately suppress the rough eye phenotype of GMR-Rac, opposing the effect of DMYPT mutants (Fig. 7 compare B with D, E and F). This suggests that the RhoA-Zip signaling pathway functions downstream of Rac, and that DMYPT is a negative regulator of the pathway.
Importantly, replacing the phosphorylation sites of Sqh with alanine remarkably suppressed the rough eye phenotype, while replacing them with glutamic acid to mimic phosphorylation slightly enhanced the phenotype (Fig. 7 compare B with H, Fig. 8 compare D with E and F). This suggests that dephosphorylation of Sqh is important in eye morphogenesis and that DMYPT may be involved in regulating the dephosphorylation of myosin light chain in eye development.
To examine whether other myosins are also involved in this process, we
tested the effect of myosin VIIA, an unconventional myosin encoded by
crinkled (ck), in the same assay. Myosin VIIA was chosen
because ck and zip behave antagonistically in wing hair
number determination in the Drosophila adult wing
(Winter et al., 2001).
Interestingly, ck behaves oppositely to myosin II (Zip) during eye
morphogenesis since a reduction in ck activity enhances the
GMR-Rac rough eye phenotype, nearly to the same extent as a reduction
in DMYPT (Fig. 7 compare B and
G).
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DISCUSSION |
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Cell movement during dorsal closure
Several lines of evidence suggest that DMYPT is essential for
dorsal closure. First, the DMYPT03802 P-element, which
disrupts DMYPT activity, leads to embryos with a dorsal open
phenotype that can be reverted by precise excision of the insertion. Second,
deficiencies of the DMYPT locus, as well as a number of imprecise
excisions of the P-element insertions in DMYPT, are also associated
with embryonic lethality and dorsal closure defects. Third, the lethality
associated with DMYPT03802 is rescued to adulthood using a
DMYPT transgene.
Given that RhoA and zip are required for dorsal closure
(Strutt et al., 1997;
Young et al., 1993
), it is not
surprising that DMYPT, a regulator of myosin function presumed to act
downstream of RhoA, is also implicated in dorsal closure. Like RhoA
(Lu and Settleman, 1999
;
Magie et al., 1999
),
DMYPT mutations do not affect dpp expression suggesting that
the failure of dorsal closure in DMYPT mutants is independent of JNK
signaling. Nonetheless, it is somewhat unexpected that the loss-of-function
mutants of both zip and its putative negative regulator,
DMYPT, have similar rather than opposite phenotypes, each displaying
late defects in cell shape and elongation
(Young et al., 1993
). One
possibility is that the activity of myosin II has to be regulated spatially.
Sqh is phosphorylated at the leading edge, indicating activation of Zip at
that site (Mizuno et al.,
2002
). In the DMYPT mutant, in addition to the leading
edge, phosphorylated Sqh is also localized to the dorsal boundaries of the
leading edge cells. Thus this pool of mislocalized active Sqh may increase the
activity of Zip where it is normally less active, ultimately interfering with
dorsal cell movement. Another possibility is that dynamic regulation of Zip
activity is important for cell sheet movement. Perhaps activation of Zip is
required for cell shape changes in the ectoderm and for maintaining tension as
the epithelial front moves forward, but concomitant inactivation of Zip is
also necessary for the cells to modulate adhesion allowing forward motility.
This paradoxical requirement of myosin II activity is similar to the function
of cell adhesion in cell movement; some cell adhesion is necessary for cell
movement, but strong adhesion inhibits cell movement.
Role of DMYPT in ring canal growth during oogenesis
Drosophila oogenesis starts with cystoblasts undergoing 4 rounds
of cell division. Through an unknown mechanism, cytokinesis of the cyst cell
is arrested and the cleavage furrow that separates the cells does not close
completely. The cleavage furrow is then stabilized and transformed into an
early ring canal, which contains only an outer rim including the actin binding
protein anillin (Field and Alberts,
1995), glycoprotein mucin-D
(Kramerova and Kramerov,
1999
), and phosphotyrosine proteins
(Robinson et al., 1994
). Then,
filamin (cheerio) (Li et al.,
1999
; Robinson et al.,
1997
; Sokol and Cooley,
1999
), aducin-like protein hts-RC and filamentous actin are
recruited to the ring canal to form an inner-rim
(Robinson et al., 1994
;
Yue and Spradling, 1992
). At
the same time, phosphotyrosine proteins are also detected in the inner rim.
Src64 and Tec29 are responsible for most of the phosphotyrosine staining
(Dodson et al., 1998
;
Guarnieri et al., 1998
;
Roulier et al., 1998
). Later,
the inner rim is further stabilized by the actin bundling protein kelch
(Kelso et al., 2002
;
Xue and Cooley, 1993
).
Ring canals grow in diameter, thickness and length in two phases
(Tilney et al., 1996). First,
the canal increases in thickness (
6 fold) from stage 2 to 5, while its
diameter and length barely grow. At the same time the number of actin
filaments increase from 80 at stage 2 to
700 at stage 6. Second, the
diameter and length grow enormously, while the thickness stays the same.
During the second phase, the actin filaments are changed into discrete
bundles. Astonishingly, the total number and density (number of filaments per
cm2) of actin filaments remain the same.
Fluorescence recovery after photobleaching experiments have shown that the
ring canal actin is highly dynamic, constantly cycling between polymerization
and depolymerization (Kelso et al.,
2002). This, together with the involvement of the actin-nucleating
protein complex Arp2/3 in ring canal growth
(Hudson and Cooley, 2002
),
argues that ring canals grow by de novo actin polymerization and regulated
cross-linking. This model requires that the newly assembled actin filaments
must slide past other bundles since there is no seam in the ring canal.
DMYPT could function at several times during ring canal formation,
including cytokinesis arrest, initiation of ring canal formation, or growth of
the ring canal. Since the ring canal starts as a cleavage furrow of
cytokinesis, myosin II is presumably there. DMYPT may be necessary to inhibit
myosin-powered contraction because in the DMYPT mutant we observe
that the ring canals are smaller, presumably as a result of overcontraction.
Secondly, the sliding of the anti-parallel actin filaments is likely to be
driven by myosin. In the absence of myosin activity, such as in the
sqh mutant GLC, ring canals are deformed, often not smooth and round,
but pointed, and loosely packed (Jordan
and Karess, 1997). The deformed ring canals may also contain
seams. However, as during dorsal closure, the activity of myosin must be
precisely regulated. Unregulated myosin II activity, for example, in the
DMYPT mutant, may cause over-sliding of the actin filaments, thus
blocking ring growth, while maintaining overall ring canal morphology.
Finally, myosin II may be involved in actin filament turnover or bundling. In
this case, hyperactivated myosin in the DMYPT mutant may cause the
actin filaments to be constitutively locked, unable to incorporate new actin
to promote ring canal expansion.
Loss of sqh activity also blocks dumping of the nurse cell
cytoplasm into the oocyte (Wheatley et
al., 1995). This is related in part to the inactivity of myosin II
in the cell cortex in sqh mutants, which under normal circumstances
provides the force for rapid transport of nurse cell contents to the oocyte
during dumping. Currently from our analysis of DMYPT GLCs, it is not
clear whether DMYPT also regulates the activity of zip in
the cell cortex.
Mutations in sqh have also pinpointed roles of myosin II in
morphogenesis of interfollicular stalks, border cell migration, centripetal
cell ingression, and dorsal appendage cell migration, all processes that
involve the somatic tissue surrounding the germline during oogenesis
(Edwards and Kiehart, 1996).
Centripetal cell ingression has been compared to dorsal closure because myosin
II is highly localized and forms a ring at the leading edge of the migrating
cells like those at the leading edge of the ectoderm during dorsal closure. It
will be very intriguing to see if DMYPT has functions in these
somatic cells of the egg chamber.
Regulation of myosin II
The regulation of MRLC phosphorylation is essential to modulate myosin II
activity and can be controled by several distinct mechanisms. For instance,
RhoA can activate its effector ROCK that in turn phosphorylates MYPT, either
directly or indirectly. MYPT phosphorylation inhibits the phosphatase activity
of MLCP and leads to elevation of MRLC phosphorylation. Phosphorylation of
MRLC can also be increased by activation of MLCK, another downstream target of
RhoA (reviewed by Hartshorne et al.,
1998; Somlyo and Somlyo,
2000
). Thus, the antagonistic activity of kinase and phosphatase
is thought to engender a delicate balance of myosin II activity modulated
through the phosphorylation state of its regulatory light chain.
To assess the relationship between DMYPT regulation of myosin II and
signaling via the Rho GTPase family members, we turned to the
Drosophila eye where sensitive genetic interactions can be observed.
One study has implicated RhoA function downstream of, or in parallel
with, Rac during orientation of ommatidia in the eye
(Fanto et al., 2000).
Consistent with this, we found that reducing the amount of RhoA, Drok
and zip partially alleviates the eye defect associated with
overexpression of Rac, while reducing the dosage of a putative negative
regulator of myosin enhances the rough eye phenotype. Furthermore, expression
of a non-phosphorylatable form of Sqh, which presumably reduces the
activity of Zip, dramatically rescues the phenotype, while overexpression of a
phospho-mimicking Sqh mutant, which should increase the activity of myosin,
exacerbates the eye defects. Taken together, these data indicate that the
regulation of myosin II activity via balancing the phosphorylation level of
Sqh is critical for proper morphogenesis of the Drosophila eye. Based
on our results, we propose that it is DMYPT that mediates myosin II
downregulation in this system.
Recently, Winter and colleagues have identified similar genetic
interactions between RhoA, Drok and zip in restricting the
number of F-actin based prehairs in the development of wing cells
(Winter et al., 2001). Not
surprisingly, the same genetic relationship holds true during dorsal closure
(Mizuno et al., 2002
).
Overexpression of Drok, a positive regulator of Zip, phenocopies a
mutation in the negative regulator, DMYPT. Moreover, a
loss-of-function mutation of zip potently suppresses the embryonic
lethality caused by mutation of DMYPT or over expression of
Drok. In other developmental contexts, myosin II functions downstream
of Rho and/or MYPT
(Halsell et al., 2000
;
Mizuno et al., 2002
;
Piekny et al., 2000
)
suggesting that similar mechanisms underlie all of these very diverse
biological processes. Since they all require actin cytoskeletal
reorganization, it suggests that RhoA regulates cytoskeletal remodeling in
non-muscle cells in vivo through the RhoA kinase-MYPT-myosin II pathway.
Interestingly, crinkled (myosin VIIA), an unconventional myosin,
behaves antagonistically to Zip/myosin II in both eye morphogenesis (this
study) and wing hair number restriction
(Winter et al., 2001). This
suggests that various myosins interact in different cell types to regulate
reorganization of the actin cytoskeleton. It will be interesting to determine
the specificity of functions of different myosins and their modes of
regulation. Since there are many different myosins, and yet a single MYPT in
Drosophila, it remains to be determined whether, and how, DMYPT
interacts with other myosins.
In conclusion, we have identified the Drosophila homologue of mammalian MYPT, named DMYPT accordingly. DMYPT plays multiple roles during Drosophila development. Loss of DMYPT function leads to blockage of rapid transport of nurse cell cytoplasm, inhibition of ring canal growth, failure of dorsal closure, defects of eye morphogenesis, and other unidentified processes during pupae development. Furthermore, our data indicate that dynamic regulation of myosin II activity via regulating phosphorylation level of myosin regulatory light chain by DMYPT is critical for the function of myosin II.
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ACKNOWLEDGMENTS |
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