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Copyright © 2002, The American Society for Cell Biology Conserved Domains of the Nullo Protein Required for Cell-Surface
Localization and Formation of Adherens Junctions Judith Kimble, Monitoring Editor ‡Corresponding author. E-mail address:
ewieschaus/at/molbio.princeton.edu. Received August 22, 2001; Revised October 10, 2001; Accepted October 12, 2001. ![]() | |||||||||||||
Abstract During cellularization, the Drosophila melanogaster
embryo undergoes a transition from syncytial to cellular blastoderm
with the de novo generation of a polarized epithelial sheet in the
cortex of the embryo. This process couples cytokinesis with the
establishment of apical, basal, and lateral membrane domains that are
separated by two spatially distinct adherens-type junctions. In
nullo mutant embryos, basal junctions fail to form at
the onset of cellularization, leading to the failure of cleavage furrow
invagination and the generation of multinucleate cells. Nullo is a
novel protein that appears to stabilize the initial accumulation of
cadherins and catenins as they form a mature basal junction. In this
article we characterize a nullo homologue from D.
virilis and identify conserved domains of Nullo that are
required for basal junction formation. We also demonstrate that Nullo
is a myristoylprotein and that the myristate group acts in conjunction
with a cluster of basic amino acids to target Nullo to the plasma
membrane. The membrane association of Nullo is required in vivo for its
role in basal junction formation and for its ability to block apical
junction formation when ectopically expressed during late
cellularization. | |||||||||||||
INTRODUCTION Drosophila cellularization is a
large-scale cytokinetic event in which thousands of syncytial nuclei
are simultaneously packaged into individual cells. Cleavage furrows
extend from the embryonic surface between neighboring nuclei, rapidly
generating a polarized epithelial sheet (reviewed in Knoblich, 2000 The patterns of synchronous nuclear division and migration during the
13 division cycles preceding cellularization lead to the formation of a
cortical monolayer of nuclei just beneath the embryonic surface. A
bulge of plasma membrane, or somatic bud, forms above each nucleus
within the cortical array (Foe and Alberts, 1983 nullo is one of three zygotic genes specifically required
for organization of the cellularization front in the
Drosophila embryo (Merrill et al., 1988 Cloning of the nullo gene revealed little about potential
mechanisms for its involvement in basal junction formation:
nullo encodes a small, highly basic protein that lacks
homology to any previously characterized proteins (Rose and Wieschaus,
1992 | |||||||||||||
MATERIALS AND METHODS Fly Stocks nullo mutants were obtained from the lines
Df(1)6F1-2, Df(1)LVII9, Df(1)LV16 (16.3), Df(1)L-II-27–32 R5 (Rose and
Wieschaus, 1992 ![]() Quantitation of Nullo Activity To quantitate the severity of the nullo phenotype,
Df(1)6F1-2 embryos were raised at 18°C, 25°C, or 29°C, and the
embryos were stained using anti-Arm to visualize cell outlines and
Hoechst to visualize nuclei (see below). A region containing an average
of 143 nuclei was examined in 20 embryos at each temperature to
determine the percentage of nuclei in multinucleate cells.Transgenes were assayed for adult rescue activity by crossing w; P[nullo]/P[nullo] males to Df(1)6F1-2/FM7 females and scoring the number of surviving Df(1)6F1-2/Y; p[nullo ]/+ offspring as a percentage of the Df(1)6F1-2/+; p[nullo ]/+ offspring. These crosses were done at 18°C, 25°C, and 29°C using multiple lines carrying each transgene. Rescue at 18°C is reported relative to the background survival of Df(1)6F1-2/FM7 females crossed to males lacking a transgene. To measure transgene rescue of the nullo phenotype, embryos from C(1)DX y w; P[nullo]/P[nullo] lines raised at 25°C were stained to visualize cell outlines and nuclei (see below). The percentage of embryos displaying a mutant phenotype was determined and a rescue efficiency was calculated based on the expected 25% mutant embryos produced by the C(1)DX y w stock. To assay the activity of UAS-nullo deletion transgenes, males carrying the UAS transgene over a balancer were crossed to mat67.15 driver females, and the progeny were raised at 18°C. The percentage lethality is given as 1 − (surviving transgene flies/surviving balancer flies). For transgenes that rescued the nullo phenotype, subcellular localization was examined in Df(1)6F1-2/Df(1)LVII9; p[nullo]/p[nullo] lines. Both Df(1)6F1-2 and Df(1)LVII9 contain additional lethal mutations, so the transheterozygote was produced to make a stock that would be viable in the presence of the nullo transgene. Nonrescuing transgenes were examined in nullo-X embryos produced by the C(1)DX y w; P[nullo]/P[nullo] stock. DV nullo Cloning A genomic D. virilis λ phage library (kindly
provided by P. Schedl) was plated and screened with a
32P-labled D. melanogaster
nullo cDNA clone (Rose and Wieschaus, 1992 ![]() ![]() Histology In situ hybridization to D. virilis embryos was done
by standard methods (Tautz and Pfeifle, 1989 ![]() To visualize Armadillo or Nullo proteins, embryos were heat-methanol
fixed (Müller and Wieschaus, 1996 To label Golgi vesicles, embryos were fixed in 18.5% formaldehyde
saturated with heptane, methanol-popped, and stained using mouse
anti–β-COP antibodies (gift of V. Malhotra; Ripoche et
al., 1994 For actin staining, embryos were fixed in 18.5% formaldehyde saturated with heptane, manually devitellinized, and treated with Alexa 488 phalloidin. Alexa 568–labeled antibodies (Molecular Probes, Eugene, OR) were used for single stainings, and a combination of Alexa 546– and Alexa 488–labeled secondaries (Molecular Probes) were used for double labeling. All embryos were stained with Hoechst, mounted in Aquapolymount (Polysciences, Warrington, PA), and imaged using a Zeiss LSM-510 confocal microscope (Thornwood, NY). Deletion Constructs Rescue assays were used to identify a 2.2-kb PstI
DM nullo genomic region that was sufficient to rescue the
nullo phenotype. This fragment was subcloned into pBS+
(Promega, Madison, WI), and an open reading frame (ORF) cassette was
created by introducing an NdeI site at the initiator
methionine and a BglII site downstream of the stop codon
using the Altered Sites Mutagenesis System (Promega). These changes did
not affect the rescue activity of the DM nullo fragment.The construct DMDV was produced by PCR amplification of the DV nullo gene using primers containing NdeI and BglII sites. The resulting DV nullo ORF cassette was then placed in the 2.2-kb DM nullo genomic fragment. The ΔM mutation was created using site-directed mutagenesis to change
the start of the DM nullo protein from MGSTHS to MASTHS.
ΔE was created by amplifying a fragment of DM nullo
corresponding to amino acids 169–213 using standard PCR. The remaining
constructs were created using PCR gene SOEing (Vallejo et
al., 1995 All of the PCR products were subcloned into the NdeI and
BglII sites of the cassette vector using NdeI and
BglII sites on the external PCR primers. The 2.2-kb
PstI nullo genomic fragment was then subcloned
into the CaSpeR-4 transformation vector (Thummel and Pirotta, 1991 The UAS full-length nullo construct was created by placing a
PstI fragment from nullo-HA (Hunter and
Wieschaus, 2000 Germline transformation was carried out by standard methods (Spradling,
1986 Western Blots Extracts for Western blots were made from heat-methanol–fixed
blastoderm embryos. The embryo extracts were run on a 12% Tris-glycine
SDS-polyacrylamide gel, and proteins were transferred onto a Hybond-P
PVDF membrane (Amersham, Arlington Heights, IL). Nullo protein was
detected using a 1:10 dilution of the 5C3-12 antibody. The ΔE protein
was detected using the 2F8-18 antibody. α-Tubulin was detected using
a 1:1000 dilution of mouse anti–α-tubulin (Sigma, St. Louis, MO).
Peroxidase-labeled horse anti-mouse (Vector Laboratories, Burlingame,
CA) was used as a secondary antibody, and protein detection was carried
out with Renaissance Chemiluminescence Reagents (New England Nuclear,
Boston, MA).To quantitate UAS-Nullo expression, Western blot analysis was carried out as above, except that detection was performed using the ECL Plus kit (Amersham). The membrane was exposed to Hyperfilm ECL (Amersham) and then scanned and analyzed using the Storm imaging system (Molecular Dynamics, Sunnyvale, CA). NMT Assay nullo ORFs were mutated as described above and
subcloned into the NdeI and BglII sites of the
pRSETA (Invitrogen). BL21(DE3) Escherichia coli strains were
cotransformed with the pRSETA construct and the pBB131 yeast N-terminal
myristoyltransferase plasmid (gift of J. Gordon), and both proteins
were induced with 1 mM IPTG. The N-myristoylation assay was carried out
as described (Duronio et al., 1990 ![]() | |||||||||||||
RESULTS Low Temperatures Compensate for the Absence of Nullo Although Nullo is required for the rapid formation of basal
junctions at the onset of cellularization, it does not appear to be an
essential component of the basal junction. In wild-type embryos, Nullo
protein is undetectable by late cellularization, whereas basal
junctions persist until the onset of gastrulation. In addition, embryos
completely lacking Nullo protein continue to form some functional basal
junctions, depending on the genetic background and environmental
conditions (Simpson and Wieschaus, 1990 ![]() ![]() Raising the LII27.32R5 stock at 18°C increased the proportion of viable nullo males. To determine if temperature would also affect the viability of nullo null mutations we examined a line transheterozygous for Df(1)6F1-2 and Df(1)LVII9, two small deficiencies that delete the nullo gene. Although this line produces no viable adults at 25°C, we could recover up to 10% of the nullo mutant class as viable adults at 18°C. To quantitate the effect of temperature on basal junction formation we examined Df(1) 6F1-2 embryos at 29°C, 25°C, or 18°C and determined the percentage of nuclei in multinucleate cells (Figure 1A). Wild-type embryos do not produce multinucleate cells at any of the temperatures tested. In contrast, at 29°C the majority of nullo mutant embryos had >80% of their nuclei in multinucleate cells (Figure 1, A and D). At 25°C, nullo mutant embryos had 50–80% of their nuclei in multinucleate cells. When the temperature was lowered to 18°C, there was a further reduction in the number of defective cleavage furrows: most embryos had <30% of their nuclei in multinucleate cells, and these cells were generally bi- or trinucleate (Figure 1, A and C). In addition, only 17% of the embryos had a nullo phenotype, as determined by anti-Arm staining of basal junctions, suggesting that the remaining 8% (of an expected 25%) of nullo mutant embryos were phenotypically wild type. This class may correspond to the viable mutant adults recovered at 18°C. Thus, reducing the temperature can compensate for the absence of Nullo protein and increase the number of functional basal junctions.
In wild-type embryos, Nullo is required to maintain the initial accumulation of basal junction components, allowing for the rapid construction of a stable junction before the onset of cleavage furrow invagination. This function may be more important when the nascent complexes are destabilized by high temperatures. In contrast, embryos raised at low temperatures may have an inherent stability of the immature junctions, which allows them to form in the absence of Nullo. The D. virilis nullo Homologue The nullo gene encodes a small novel protein,
with no motifs or homologies to suggest a mechanism for its involvement
in basal junction formation (Rose and Wieschaus, 1992 ![]() ![]() ![]() ![]()
Like DM nullo, DV nullo is an intronless gene that encodes a small basic protein with a predicted size of 233 amino acids and a calculated pI of 10.8. The amino acid sequences of DM Nullo and DV Nullo are 47% identical, and the similarity of the proteins rises to 58% when conservative amino acid changes are included. Most of the conserved residues fall into clusters (Figure 2, A–E) in the C-terminal two thirds of the protein (Figure 2G). The N-terminal sequence of the proteins is comparatively unconserved, although it contains an N-terminal myristoylation site (M) and a cluster of positively charged residues (P) that are present in both proteins (Figure 2G). We were interested in determining if the conservation between the two proteins was sufficient to allow rescue of D. melanogaster nullo mutant embryos using the DV nullo gene. We created transgenic lines that contained the D. virilis nullo ORF under the control of the D. melanogaster nullo promoter (DMDV). To assay the rescue of mild, moderate, and severe cellularization defects, we tested the ability of the transgene to restore adult viability to nullo mutants raised at 18°C, 25°C, or 29°C. A single copy of the DMDV transgene was sufficient to rescue mutant flies at all three temperatures (Figure 3A). To assess rescue of the cellularization defects directly, we examined embryos from a nullo mutant line that carried two copies of the DMDV transgene. At 25°C none of the embryos from this line had a detectable nullo phenotype, suggesting the transgene completely restores basal junction formation (Figure 3B).
Transgenes containing a D. virilis genomic fragment are also
capable of rescuing D. melanogaster nullo mutant embryos,
although the rescue activity is lower than constructs containing the
D. melanogaster promoter region and the D.
virilis protein coding region (our unpublished results). This
would suggest that there is at least partial conservation of the
regulatory regions that drive blastoderm-specific expression. These
regions are likely to be contained within a small upstream region,
because the D. virilis fragment contains only a limited
region of upstream DNA. We have also observed that a D.
melanogaster nullo transgene containing only 400 base pairs of
upstream DNA is capable of reproducing the short burst of
nullo expression, although the anterior-posterior striping
seen during nullo degradation is less pronounced (our
unpublished results). Interestingly, this small upstream region
includes a regulatory motif shown to be conserved between DM
nullo and sry-α, another zygotic
cellularization gene with a similar pattern of expression (Ibnsouda
et al., 1993 The conservation of sequence and expression pattern, along with the ability to perform interspecies rescue, confirm that the D. virilis gene identified is a structural and functional homologue of D. melanogaster nullo. The rescue experiments also identify a set of conserved features that are sufficient for Nullo activity during cellularization. Identification of Conserved Regions Required for Nullo Function The majority of the conserved amino acids are grouped into five
clusters (A–E) in the C-terminal region of the Nullo protein. To
assess the role of this conservation, we created a series of DM Nullo
transgenes (ΔA–ΔE), each lacking a single conserved region. We
then introduced these transgenes into nullo mutant lines,
examined the expression and subcellular localization of the mutant
proteins, and assayed the ability to these transgenes to rescue the
nullo mutant phenotype.Mutations ΔA-ΔD did not affect the level of Nullo protein expression: extracts from the transgenic embryos contained truncated proteins of the predicted size at levels comparable to wild type (Figure 4A). Surprisingly, deletion of these conserved regions had no effect on the subcellular localization of the Nullo protein. These deletion proteins accumulated at the cellularization front and showed normal punctate staining in the cortical cytoplasm (Figure 4B). ΔE deleted the epitope for the 5C3-12 anti-Nullo antibody, and although we were able to use an alternative mAb to demonstrate that this protein is produced at reduced levels, we were only able to detect faint staining in the embryo (Figure 4, A and B).
Adult rescue assays and the subsequent examination of nullo mutant embryos carrying two copies of each transgene revealed that ΔC and ΔD were capable of rescuing the nullo phenotype at 18°C, 25°C, and 29°C, whereas ΔA, ΔB, and ΔE failed to rescue at any temperature (Figure 3, A and B). The fact that ΔA and ΔB produce wild-type levels of Nullo protein, which is enriched at the cellularization front yet fail to rescue the nullo phenotype, suggests that these conserved regions are specifically required for Nullo to stabilize the accumulation of basal junction components in the nascent cleavage furrows. Nullo Is Modified by N-terminal Myristoylation Although the N-termini of the DM Nullo and DV Nullo proteins have
only a limited degree of amino-acid conservation, they do contain two
conserved motifs: a consensus site for N-terminal myristoylation and a
cluster of positively charged amino acids. These sequences often act in
concert to target proteins to the plasma membrane (reviewed in Resh,
1999 ![]() To determine if DM Nullo is a substrate for N-terminal
myristoylation we expressed the nullo gene in a bacterial
strain carrying the gene for yeast N-terminal myristoyltransferase
(NMT) and grew the strains in the presence of
3H-myristate (Duronio et al., 1990
These data indicate that the Nullo protein contains a functional
N-terminal myristoylation site. Because the mechanism for N-terminal
myristoylation appears to be conserved in Drosophila
(Deichaite et al., 1988 N-terminal Myristoylation Is Required for Nullo Localization To determine if the N-terminal motifs play a role in Nullo
localization, we used site-directed mutagenesis to create three
DM nullo transgenes: the first contains the Gly2 to Ala
mutation that disrupts the myristoylation site (ΔM), the second has a
series of point mutations that neutralize the positively charged
cluster (ΔP), and the third combines these changes (ΔMP). Western
blots of extracts from the transgenic embryos show that all three
constructs produced protein at levels comparable to the endogenous
Nullo protein (Figure 5B).Although expressed at normal levels, each of these mutations caused
specific disruptions in the subcellular localization of the Nullo
protein. The wild-type Nullo protein begins to accumulate during
nuclear division cycle 12 and localizes to the metaphase furrows during
mitosis of cycle 13. At the start of cycle 14, Nullo protein localizes
to the cell surface and then becomes enriched at the cellularization
front and in punctate cytoplasmic structures (Figure 5C; Postner and
Wieschaus, 1994 Examination of ΔM revealed that, in the absence of the myristoylation site, Nullo has a normal localization to the pseudocleavage furrows during mitosis of cycle 13. However, at the onset of cellularization, Nullo adopts a nuclear localization (Figure 5C) that persists until midcellularization, when levels of localized protein decline abruptly. ΔP greatly reduces level of punctate cytoplasmic staining but has only a mild effect on membrane localization (Figure 5C). This suggests that association with the punctate cytoplasmic structures is not a prerequisite for membrane localization. The loss of both the myristoylation site and positive charge completely blocks Nullo protein localization: ΔMP is distributed diffusely throughout the cortex of the embryo (Figure 5C). These results suggest that the combination of conserved N-terminal motifs is essential for targeting Nullo protein to the cellularization front. We then examined the ability of these mutant transgenes to rescue the nullo phenotype. We found that a single copy of either ΔM or ΔP was sufficient to rescue nullo flies to adult viability at 18°C, 25°C, or 29°C (Figure 3A) and that embryos with two copies of ΔM or ΔP have a normal hexagonal array at 25°C (Figure 3B). A single copy of ΔMP, however, partially restored viability only at 18°C. Two copies of ΔMP were capable of restoring a normal hexagonal array to 49–83% of embryos at 25°C, depending on the transgene line tested. Although ΔMP is not enriched at the plasma membrane, some portion of this protein may still reach the cellularization front. As with the hypomorph LII27.32R5, this low level of Nullo protein may increase the likelihood of basal junction formation. N-terminal Motifs Are Required for the Localization of Ectopic
Nullo during Late Cellularization The wild-type Nullo protein is rapidly degraded before
the completion of cellularization and the formation of the apical spot
junctions (Postner and Wieschaus, 1994 ![]() ![]() ![]() ![]() Prolonging the expression of full-length Nullo at levels characteristic of early cycle 14 is sufficient to produce the ectopic phenotype. To ensure that the potential absence of an ectopic phenotype in embryos from UAS-ΔM, UAS-ΔP, or UAS-ΔMP lines was not due to subnormal protein levels, the transgene insertions were first evaluated for protein expression levels. For each construct, several transgene insertions were analyzed by Western blot, Nullo expression levels were quantitated, and transgene insertions were assigned relative expression values. We then examined the subcellular localization of transgene insertions with similar expression levels. When expressed during late cellularization, Nullo protein accumulates
along the entire plasma membrane and in punctate structures in the
basal region of the newly formed epithelial cells (Figure
6; Hunter and Wieschaus, 2000
The activity of a given mutant transgene was assayed by measuring the degree of lethality caused by its ectopic expression. Using the full-length transgene to elevate Nullo protein levels during late cellularization to 69% of that observed at the beginning of cycle 14 caused lethality in 87% of embryos and blocked the formation of apical junctions (Figure 6). UAS-ΔP at similar levels caused lethality in only 43% of embryos and only resulted in a partial blockage of apical junction formation (Figure 6). UAS-ΔP therefore shows a reduction in activity but can still interfere with apical junction formation. The ectopic expression of UAS-ΔM and UAS-ΔMP, on the other hand, has no effect on viability and does not block apical junction formation (Figure 6). The failure of these mutant proteins to localize to the cell surface suggests that membrane localization is absolutely required for Nullo's interference with apical junction formation. | |||||||||||||
DISCUSSION Comparison of D. melanogaster and D.
virilis Sequences Identifies Conserved Regions of Nullo Before the cloning of DV Nullo, little was known about the aspects
of the Nullo protein involved in subcellular localization and basal
junction formation. No Nullo point mutations had been isolated in
screens for embryonic-lethal mutations on the X-chromosome (Nicklas and
Cline, 1983 ![]() ![]() ![]() Like DM nullo, DV nullo appears to require a
relatively small region of upstream DNA for proper expression. The DV
nullo gene also lacks introns, a characteristic of DM
nullo and the other zygotic cellularization genes
bottleneck and serendipity-α (Vincent et
al., 1985 Comparison of the DM Nullo and DV Nullo predicted proteins revealed
several interesting features. The first of these is the conservation of
the N-terminal myristoylation site and the presence of an adjacent
unconserved, but highly basic, region. This suggested that the
N-terminal myristoylation site was important for Nullo localization or
function and that it likely acted as part of a “myristate plus
basic” motif used in membrane localization (see below). The second
feature was the presence of five distinct conserved domains, separated
by short nonconserved stretches. This pattern of conservation is
evident even among closely related Drosophila species. In
the nullo genes of D. oreana, D.
lutescens, and D. yakuba (Caccone et al.,
1996 By deleting single conserved regions we identified two regions that affect Nullo activity without affecting protein localization. These proteins contain all the domains required not only to reach the plasma membrane but also to become enriched at the cellularization front. However, they still fail to rescue the basal junction phenotype, suggesting that they may define regions of Nullo that interact with components of the junction itself. Although the novel sequence of these regions makes it difficult to draw conclusions about their specific role, they provide a basis for further analysis, including the identification of interacting proteins. Two other conserved regions can be removed without affecting Nullo localization or activity, suggesting that they are dispensable or that they provide a redundant function. Truncation of the C terminus reduced protein levels, suggesting this region cannot be removed without affecting the stability of the Nullo protein. In addition to identifying functionally important regions, the deletion analysis also shed light on the previous difficulties in isolating point mutations in nullo. Given the small size of the nullo gene, the lack of conservation in the N-terminal third of the protein and the ability to delete substantial regions of the C terminus without affecting function, the probability of isolating a lethal point-mutation in nullo would be quite small. N-terminal Motifs Control the Localization of Nullo Protein Unlike the C terminus, which has obvious regions of sequence
conservation, the N-terminus of Nullo is highly divergent.
Nevertheless, it contains two motifs, an N-terminal myristoylation site
and a positively charged cluster, which we have shown to be essential
for its localization to the cellularization front. To give rise to a
stable association, the weak hydrophobic interactions between a
myristate group and the plasma membrane are known to require a second
contact, such as the electrostatic interaction between a basic cluster
and acidic phospholipids (reviewed in Resh, 1999 ![]() ![]() ![]() ![]() Nullo is one of ~35 sequences in the D. melanogaster
genome that contains the MGXXXS/T consensus site for N-terminal
myristoylation. Although the majority of these genes have been
characterized genetically and developmentally, only a handful have
actually been shown to be myristoylated (Neel and Young, 1994 During metaphase of nuclear cycle 13, Nullo lacking a myristoylation site (ΔM) still shows normal localization to the plasma membrane of the metaphase furrows. However, at the onset of cycle 14, ΔM is lost from the cellularization front and begins to accumulate in the nucleus where it remains until midcellularization. UAS-ΔM expressed during late cellularization showed no membrane association, but was observed in the nucleus. The difference in ΔM localization before and after the onset of cellularization suggests that some aspect of Nullo targeting or membrane association changes at this point. The fact that ΔMP, which lacks both N-terminal domains, shows no early membrane association suggests that the initial association of Nullo with the plasma membrane requires the presence of a positive charge and that the positive charge is no longer sufficient for membrane localization after the onset of cellularization. The electrostatic interaction may be disrupted by changes in the phospholipid content of the apical membrane as new membrane is inserted during cellularization. In contrast, endogenous and ectopic expression of ΔP demonstrates that the myristoylation site alone is sufficient for partial membrane localization throughout cellularization. After cycle 13, ΔM accumulates in the nucleus, which is surprising,
because Nullo does not contain a predicted nuclear localization signal,
and we have never observed wild-type Nullo protein in the nucleus. It
is possible, however that the basic cluster confers a nuclear
localization that is normally overridden by the membrane targeting of
the N-terminal myristoyl group. In the absence of the myristoyl group,
Nullo would associate with the nucleus via the polybasic region, and as
we observed, the association with the nucleus would be diminished by
the removal of the positively charged cluster (ΔMP). A similar
observation has been made with the Moloney MuLV Gag protein, where
mutation of the N-terminal myristoylation site roughly doubles the
amount of protein found in the nucleus (Nash et al., 1993 Although the ΔM, ΔP, and ΔMP proteins all retained some ability to rescue the nullo mutant phenotype, only ΔP was able to partially block apical junction formation when expressed ectopically during late cellularization. One explanation for the difference in activity may lie in the levels of functional protein required in each of these assays. As seen with the LII27.32R5 allele, the rescue of the nullo phenotype requires levels of Nullo protein much lower than those normally seen at the start of cycle 14. In contrast, the ectopic effects on apical junction formation require at least half the protein level seen at the onset of cellularization. In the case of ΔM the difference in activity may also result from the fact that this protein retains membrane localization during the cycle 13 to cycle 14 transition when basal junctions are formed but lacks any detectable membrane localization when ectopically expressed during late cellularization. The fact that both assays showed a decline in Nullo activity that correlated with the loss of Nullo from the plasma membrane suggests that the “myristate plus basic” motif controls membrane localization and that the membrane association of Nullo is essential for its role in junction formation. The fact that both ΔM and ΔP retained some ability to associate
with the plasma membrane is unusual, as removal of either the myristoyl
group or the basic region is usually enough to prevent plasma membrane
association in “myristate plus basic” proteins (reviewed in Resh,
1999 Loss of Nullo has its primary effect on the formation of basal
junctions from the small points of membrane contact that arise at the
start of cellularization. The results presented in this article argue
that Nullo is not an essential component of the junction itself but is
required to stabilize nascent junctions, especially at higher
temperatures where the membrane contacts may be more fluid. Ectopic
expression of Nullo during late cellularization may also stabilize
junctional components as they reach the plasma membrane, but in this
case it would prevent the clustering required to form the apical
adherens junctions (Hunter and Wieschaus, 2000 | |||||||||||||
ACKNOWLEDGMENTS The authors thank members of the Wieschaus and Schupbach laboratories for helpful discussions; Jen Zallen, Jorg Grosshans, Jeff Thomas, and two anonymous reviewers for comments on the manuscript; Reba Samanta and Joe Goodhouse for assistance with histology and confocal microscopy; Romy Knittel for technical assistance in cloning and characterizing the D. virilis homologue; and Cynthia Hsuan-Hung for maintaining stocks. The authors are grateful to J. Gordon, V. Malhotra, P. Schedl, and D. St. Johnston for providing stocks and reagents. This work was supported by the Howard Hughes Medical Institute grant 5R37HD15587 from the National Institute of Child Health and Human Development and grant 95-00258/3 from the U.S.-Israel Binational Science Foundation. | |||||||||||||
Footnotes Article published online ahead of print. Mol. Biol. Cell
10.1091/mbc.01–08-0418. Article and publication date are at
www.molbiolcell.org/cgi/doi/10.1091/mbc.01–08-0418. | |||||||||||||
REFERENCES
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