The Wellcome Trust/Cancer Research UK Institute and the Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
Author for correspondence (e-mail:
ds139{at}mole.bio.cam.ac.uk)
Accepted 19 May 2003
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SUMMARY |
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Key words: PAR-1, Tau, Follicle cells, Drosophila
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INTRODUCTION |
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These membrane asymmetries are propagated to other cellular compartments.
In the Drosophila follicular epithelium, for example, the cortical
spectrin cytoskeleton becomes polarised into an apical domain that is composed
of ßheavy spectrin and -spectrin, and a basolateral
domain that contains ß-spectrin/
-spectrin complexes
(Lee et al., 1997
). Actin also
becomes enriched in the apical cortex of these cells, as in other epithelia
(Baum et al., 2000
;
Mooseker, 1985
). The
microtubule cytoskeleton is also polarised to form an array of very stable
microtubules (MTs) that run parallel to the apicobasal axis, with their minus
ends at the apical membrane and their plus ends oriented toward the basal
membrane (Bacallao et al.,
1989
; Bre et al.,
1990
; Clark et al.,
1997
). In mammalian cells, at least, the distinct membrane domains
are further reinforced by sorting in the secretory pathway that delivers
different sets of proteins and lipids to the apical and basolateral domains
(Keller and Simons, 1997
).
Many epithelial cells also mediate polarised transcytosis to transport
extracellular factors from one side of the epithelium to the other
(Mostov et al., 2000
).
Although the steps in the establishment of epithelial polarity are well
characterised, little is known about how the initial extracellular cues are
transduced to polarise the different components of the cell. One group of
proteins that appear to play an essential role in this process are the PAR
proteins, which were originally identified because they are required for the
anterior-posterior polarity of the C. elegans zygote
(Kemphues et al., 1988). Three
of these proteins, PAR-3, atypical Protein Kinase C (aPKC) and PAR-6, form a
conserved protein complex that localises to the anterior cortex of the one
cell zygote, where they are required for the asymmetry of the first cell
division (Etemad-Moghadam et al.,
1995
; Hung and Kemphues,
1999
; Tabuse et al.,
1998
; Watts et al.,
1996
). The Drosophila homologues of PAR-3 (Bazooka),
PAR-6 and aPKC localise to a sub-apical region in epithelial cells to define
the position of the most apical junction, the zonula adherens, and loss of any
of these proteins leads to a loss of polarity
(Kuchinke et al., 1998
;
Muller and Wieschaus, 1996
;
Petronczki and Knoblich, 2001
;
Wodarz et al., 2000
). The
complex shows a similar localisation to the most apical junction in mammalian
epithelia, in this case the tight junction, and overexpression of kinase-dead
aPKC disrupts the localisation of the tight junction proteins and causes the
mislocalisation of apical membrane proteins
(Izumi et al., 1998
;
Suzuki et al., 2001
).
The conserved serine/threonine kinase PAR-1 has also been implicated in
cell polarity in several contexts
(Böhm et al., 1997;
Guo and Kemphues, 1995
;
Shulman et al., 2000
;
Tomancak et al., 2000
). PAR-1
localises to the posterior of the C. elegans zygote in a
complementary pattern to the PAR-3/PAR-6/aPKC complex, and is required for the
asymmetric positioning of the mitotic spindle and the posterior localisation
of the P granules (Guo and Kemphues,
1995
). Drosophila PAR-1 is required for anteroposterior
polarisation of the oocyte at two stages of oogenesis. Null mutations in
par-1 block the formation of a microtubule organising centre (MTOC)
at the posterior cortex of the early oocyte, resulting in the loss of oocyte
fate (Cox et al., 2001a
;
Huynh et al., 2001b
). The
oocyte is specified normally in hypomorphic par-1 mutants, but the
repolarisation of the oocyte MT cytoskeleton that defines the anteroposterior
axis of the embryo is disrupted (Shulman
et al., 2000
; Tomancak et al.,
2000
). In mid-stage oocytes, an unidentified signal from the
posterior follicle cells induces the disassembly of the original posterior
MTOC, and leads to the formation of a new MT array, in which most MTs are
nucleated from the anterior with their plus ends extending towards the
posterior pole (Cha et al.,
2001
; Clark et al.,
1994
; Clark et al.,
1997
; Theurkauf et al.,
1992
). In par-1 mutants, MTs are found more evenly around
the oocyte cortex, and the plus ends are focussed on the centre of the oocyte
rather than the posterior (Benton et al.,
2002
; Shulman et al.,
2000
). As a consequence, oskar mRNA is mislocalised to
the middle of the oocyte, and the resulting embryos therefore lack an abdomen
and germline. Thus, the principal function of PAR-1 in Drosophila
axis formation appears to be to organise a polarised MT array.
Several results indicate that PAR-1 is also required for epithelial
polarity. A mouse PAR-1 homologue, EMK, localises to the basolateral membrane
of polarised MDCK cells, and overexpression of EMK lacking the kinase domain
causes these cells to lose their columnar morphology and be extruded from the
monolayer (Böhm et al.,
1997). PAR-1 shows a similar localisation to the basolateral
cortex of Drosophila follicle cells, and removal of PAR-1 from these
cells results in defects in epithelial organisation and the mislocalisation of
membrane proteins (Cox et al.,
2001a
; Shulman et al.,
2000
). As is the case in the oocyte, PAR-1 appears to play a
particularly important role in regulating the MT cytoskeleton in epithelial
cells, as mutant follicle cells have been reported to lack MTs
(Cox et al., 2001a
).
The mammalian PAR-1 homologues, MARK1 and MARK2, also regulate MTs, and are
thought to act by phosphorylating the microtubule associated proteins (MAPs),
Tau, MAP2 and MAP4 (Drewes et al.,
1995; Illenberger et al.,
1996
). These MAPs contain three to four copies of a conserved
MT-binding domain (MTBD), and bind along the length of MTs to stabilise and
stiffen them (Chapin and Bulinski,
1992
). Tau and MAP2 are highly expressed in neurons, and localise
to axons and dendrites, respectively, whereas MAP4 is expressed more widely
(Matus, 1991
). MARKs
phosphorylate a conserved KXGS motif in the MTBDs of these proteins, which
inhibits their binding to MTs (Drewes et
al., 1997
; Illenberger et al.,
1996
). MARK activity should therefore decrease MT density.
Consistent with this, overexpression of the MARKs leads to
hyperphosphorylation of these MAPs, and causes a breakdown of the MT
cytoskeleton (Drewes et al.,
1997
; Ebneth et al.,
1999
). Thus, the MARKs seem to have the opposite effect on MTs to
Drosophila PAR-1, which is required to maintain the MT array
(Cox et al., 2001a
). This
apparent difference between the function of the mammalian and
Drosophila kinases prompted us to analyse how Drosophila
PAR-1 regulates MT organisation in epithelial follicle cells, and to
investigate the role of Tau family of MAPs in this process.
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MATERIALS AND METHODS |
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w; FRT-G13-par-1W3/CyO
(Shulman et al., 2000)
y,w,hs-flp; FRT-G13-nlsGFP/CyO
(Luschnig et al., 2000)
w; FRT-G13-par-116 /CyO
(Cox et al., 2001a
)
w; UASp-par-1(N1S)-GFP/TM3,Sb (Huynh et
al., 2001b)
Gal4 follicle cell driver: w; E4
(Queenan et al., 1997)
w; 133.4 Nod:lacZ (Clark et al.,
1997)
w; KZ503 Kin:lacZ (Clark et al.,
1994)
w; FRT-82B-Df(3R)MR22(tau)/TM3, Ser act:GFP (this work)
w,hs-flp; FRT-82B-nlsGFP (Chou and
Perrimon, 1996).
Follicle cell clones
Follicle cell clones were generated by the FLP/FRT technique
(Chou et al., 1993;
Chou and Perrimon, 1996
),
using the FRT-G13-nlsGFP chromosome. Clones were induced by heat-shocking
third instar larvae or adult females at 37°C for 2 hours on two
consecutive days. Female were dissected 1 day after the last heat-shock.
Colcemid treatment
Flies were starved for 5 hours and then fed with 200 µg/ml colcemid
(Sigma) mixed with some dry yeast for 16 hours, and dissected immediately.
Cold shock
Females were kept on ice for 1 hour and dissected either immediately or
after a recovery time at room temperature.
Fluorescence quantification
Quantification of the intensity of the GFP and the -Tubulin staining
was measured using the Laser Pix4 software (BioRad)
(Cha et al., 2002
).
Cloning of Drosophila tau
tau cDNAs were isolated from the Berkeley Drosophila
Genome Project (BDGP) adult head (GH) ZAPII cDNA library using a PCR product
spanning the genomic region encoding the Tau MTBD as a probe, following
standard procedures.
Antibody production and western analysis
The Tau antibody was raised in rabbits against a 6xHis tagged
C-terminal fragment of Tau-A (amino acids 183-375), and affinity purified
using a purified MBP fusion of the same fragment, following standard
procedures (Harlow and Lane,
1988; Huang and Raff,
1999
). Embryo extracts for SDS-PAGE/western blotting were prepared
by boiling and homogenising 12-18 hours embryos in Laemmli sample buffer.
tau mutant generation
EP(3)3597 and EP(3)3203 were identified from the BDGP P
element disruption project collection database. To generate deletions
uncovering the tau locus, we induced P element-mediated male
recombination (Preston et al.,
1996), between the EP(3)3203 chromosome and a homologue
bearing the flanking visible markers ebony (e) and
claret (ca). From 14,000 progeny, we identified one e
ca+ recombinant chromosome (MR22), which was homozygous lethal
but retained the original EP(3)3203 insertion. Using inverse PCR, we
determined the presence of a deletion between the 5' end of
EP(3)3203 and position 144091 in genomic contig AE003761. This
deletion, Df(3R)MR22(tau), was recombined on to the FRT 82B
chromosome to generate homozygous clones.
Microtubule-spin down assay
Twelve- to 18-hour-old embryos were homogenised in an equal volume of
C-buffer (50 mM HEPES, pH 7.6, 1 mM MgCl2, 1 mM EGTA) with a
Complete Protease Inhibitor cocktail (Roche). The extract was centrifuged for
1 hour at 100,000 g. Dithiothreitol and GTP were added to the
supernatant to 1 mM final concentration, and this was split into two equal
aliquots. To one aliquot, Taxol was added to 10 µm, to polymerise the
tubulin, whereas only buffer was added to the other. The supernatants were
warmed to 25°C for 5 minutes to allow polymerisation to initiate, and then
shifted to 4°C for a further 15 minutes. The supernatants were layered
onto a 2 volume cushion of C-buffer with 50% sucrose and this was centrifuged
at 100,000 g for 10 minutes. Both supernatant and pellet were
resuspended in Laemmli sample buffer and analysed by SDS-PAGE and western
blotting.
Generation and analysis of transgenic lines
The pUASp-tau-A:mGFP6 transgene contains the full-length
tau-A ORF, lacking the STOP codon, upstream of the GFP variant,
mGFP6, in the pUASp vector (Rørth,
1998; Schuldt et al.,
1998
). Transgenic lines were generated by standard methods and
crossed to nanos-GAL4:VP16. Females were dissected in 10S Voltaleff
oil (Atochem) and viewed under an inverted confocal microscope.
Kinase assay
In vitro kinase assays were performed as previously described
(Benton et al., 2002). The
MBP:Tau-A MTBD (amino acids 144-375) substrate was expressed in and purified
from bacteria. The mutant variant (`KXGA'), containing the mutations S184A,
S243A, S275A and S305A in the four KXGS motifs, was generated by
oligonucleotide-directed mutagenesis.
Staining procedures
Females were fattened for 24 hours and the ovaries dissected in PBT (PBS +
0.1% Tween), fixed for 10 minutes or 20 minutes with 8% or 4%
paraformaldehyde/PBT respectively, washed three times for 10 minutes with PBT,
blocked with PBT-10 (PBT + 10% BSA) for 1 hour and incubated with the antibody
in PBT-1 (PBT + 1% BSA) overnight. After several washes with PBT for 2 hours,
the ovaries were incubated with the secondary antibody for 4 hours. They were
finally washed three times with PBT for 15 minutes and mounted in Vectashield
(Vector). All steps were performed at room temperature. Primary antibodies
were used as follows: mouse anti-Armadillo (7A1) (1/200)
(Riggleman et al., 1990); rat
anti-DE-Cadherin (1/2000) (Oda et
al., 1994
); mouse anti-Crumbs (cq4) (1/50)
(Tepass et al., 1990
); rabbit
anti-ß-gal (1/2000, ICN Pharm, Cappel); mouse anti-ß-PS-Integrin
CF6G11 (1/3) (Brower et al.,
1984
); mouse anti-NotchICD (C179C6) (1/1000)
(Fehon et al., 1991
); rat
anti-Neurotactin BP106 (1/40) (Hortsch et
al., 1990
); rabbit anti-nPKC (1/500, Santa Cruz Biotechnology);
rabbit anti-
-Spectrin (1/500) (Byers
et al., 1987
); rabbit anti-ß-Spectrin (1/200)
(Byers et al., 1989
); mouse
anti-ßheavy spectrin (1/200)
(Thomas and Kiehart, 1994
);
mouse anti-
-Tubulin DM1A (1/500, Sigma). FITC- and Red Texas-conjugated
secondary antibodies (Molecular Probes) were used at 1/100 dilution. Actin
staining was performed with Rhodamine-conjugated phalloidin (Molecular
Probes). All confocal micrographs were collected using a BioRad MRC1024 scan
head mounted on a Nikon E800 microscope.
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RESULTS |
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To confirm that the MTs are less stable in mutant cells, we examined their resistance to treatments that promote MT depolymerisation. The MTs in follicle cells are extraordinarily resistant to disassembly, as they are still present after more than 24 hours exposure to the MT-depolymerising drug, colcemid. By contrast, par-1 mutant follicle cells fixed under optimised conditions lack visible MTs after short colcemid treatments (Fig. 4A). The disassembly of dynamic MTs is also promoted by cold, and we therefore kept females on ice for 1 hour before fixing their ovaries with the optimised protocol. The mutant cells again appear to lack MTs, whereas the MTs in the non-mutant cells become fuzzy (Fig. 4B). This depolymerisation is reversible, because MTs reappear in mutant cells if the females are allowed to recover from the cold shock for 5 minutes (Fig. 4C). After 10 minutes recovery, mutant cells show a higher density of MTs than the adjacent heterozygous cells, as they do in the absence of cold shock (Fig. 4D). Thus, PAR-1 is required to stabilise the MTs in cells, and its removal leads to the formation of more MTs that are less stable.
In epithelial cells, most MTs exhibit a uniform apicobasal polarity, with
their minus ends localised at the apical membrane and their plus ends
extending towards the basal membrane
(Bacallao et al., 1989;
Bre et al., 1990
;
Mogensen et al., 1989
). This
is also the case in epithelial follicle cells and can be visualised using
motor proteins as markers for the plus and minus ends of MTs
(Clark et al., 1997
). A
Kin:ß-gal fusion protein containing the motor domain of the plus
end-directed MT motor, Kinesin 1, accumulates at the basal side of the cell,
whereas a Nod:ß-gal fusion protein localises apically. Nod:ß-gal
shows an identical localisation to the apical membrane in par-1
mutant follicle cells as in wild-type cells, indicating that the distribution
of minus ends is not dramatically affected
(Fig. 5A). By contrast, the
plus end marker, Kin:ß-gal, accumulates in the centre of mutant cells,
most probably around the nucleus (Fig.
5B). High magnification views of MTs in follicle cells show the
MTs extending from the apical to the basal cortex, with a lower density along
the basal membrane. By contrast, par-1 mutant follicle cells show a
high density of MTs along the basal cortex
(Fig. 5C).
|
|
To examine whether Tau is a substrate for PAR-1, we performed in vitro kinase assays, in which immunoprecipitated PAR-1 was incubated with bacterially expressed Tau in the presence of labelled ATP. PAR-1 phosphorylates Tau in this assay, but this is not significantly affected by replacing the serines with alanine in all four of the KXGS motifs within the Tau MTBD (Fig. 6F). These results indicate that, although Tau is a substrate for PAR-1 in vitro, this phosphorylation is not at the same regulatory sites as described for the MARK kinases.
To address the role of Tau in MT organisation, we sought to identify mutant alleles of the gene. Two P element insertions, EP(3)3203 and EP(3)3597, have been recovered in the first intron of the tau locus, close to the start of the S10 gene (Fig. 6A). Embryos homozygous for either insertion show no detectable Tau protein on western blots, indicating that they are both strong tau mutants, and we have therefore renamed them tauEP(3)3203 and tauEP(3)3597. Surprisingly, homozygotes of both alleles are viable and fertile, and display no obvious morphological or behavioural defects. Moreover, the organisation of MTs in both follicle and germline cells is indistinguishable from wild type (data not shown). As it is possible that the EP elements do not completely abolish Tau expression, we used transposase-mediated male recombination to generate deletions that remove the tau locus, but not S10. One recombinant, Df(3R)MR22, is a 62 kb deletion extending distally from EP(3)3203 that removes almost all of the tau locus, including the exons encoding the MTBD (Fig. 6A). Germline and follicle cell clones of Df(3R)MR22 also display no MT defects (Fig. 6G and data not shown). Consistent with this, we observed no Tau expression in the follicle cells. Thus, Tau is apparently dispensable in Drosophila and is not required for MT organisation during oogenesis.
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DISCUSSION |
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Although par-1 mutants produce similar polarity phenotypes in the
follicle cells to mutants in the components of the Bazooka/PAR-6/aPKC complex,
they are not identical. par-1 clones cause a complete disruption of
polarity only when induced early in the follicle cell lineage. The smaller
clones that arise later in oogenesis often show little or no reduction in the
localisation of apical and basolateral markers, and usually remain as a single
layer of cells. By contrast, even late clones of bazooka or
aPKC produce penetrant epithelial defects
(Cox et al., 2001b) (R.B. and
D.St.J., unpublished). This difference is also apparent in the embryo, where
loss of zygotic bazooka, PAR-6 or aPKC disrupts epithelial
organisation (Kuchinke et al.,
1998
; Muller and Wieschaus,
1996
; Petronczki and Knoblich,
2001
; Wodarz et al.,
2000
). par-1 homozygous embryos, however, display no
obvious epithelial polarity phenotype, although it is not possible to remove
the maternal PAR-1 completely (Shulman et
al., 2000
; Sun et al.,
2001
). The reason for low penetrance of polarity defects in
smaller par-1 follicle clones is unclear, but similar differences
between early large clones and smaller late clones have been observed for
crumbs, discs lost and lkb1
(Martin and St Johnston, 2003
;
Tanentzapf et al., 2000
). One
possibility is that these genes are required for the initial formation of the
follicular epithelium, but not for its maintenance. This seems unlikely to be
the case for par-1, however, because small clones containing only one
or two cells can sometimes show strong apicobasal polarity defects. A more
likely explanation is that the low penetrance of this phenotype in small
clones is due to the perdurance of the PAR-1 that was present at the time when
the clones were induced. In support of this, the penetrance of the polarity
defects of par-1 clones increases with clone size and the stage of
oogenesis, as one would expect if the protein is gradually degraded over time,
and is diluted out by cell division.
In contrast to apicobasal membrane polarity, the density, the stability and
the organisation of MTs are disrupted in all par-1 clones, regardless
of their size or the stage of oogenesis. This is likely to represent a
distinct function of the kinase from its other roles in cell polarity, because
it is much more sensitive to a reduction in activity. The effects of PAR-1 on
MT density are consistent with results on the mammalian PAR-1 homologues,
MARK1 and MARK2. Our experiments show that removal of PAR-1 causes an increase
in the density of MTs in each cell, whereas overexpression of MARK1 or MARK2
in unpolarised tissue culture cells causes most MTs to disappear
(Drewes et al., 1997;
Ebneth et al., 1999
).
The MARKs have been proposed to regulate the MT cytoskeleton by
phosphorylating Tau family MAPs, thereby inhibiting them from binding to and
stabilising MTs. Although PAR-1 does phosphorylate Drosophila Tau in
vitro, tau null mutations are viable and fully fertile and have no
effect on the arrangement of MTs in either the follicle cells or the oocyte.
Therefore, this mechanism cannot account for its function in organising the
MTs in the follicle cells. The viability of tau mutants is
surprising, given the many functions ascribed to Tau in human neurons
(Lee et al., 2001). It does
have a precedent, however, as tau knockout mice are viable, have a
morphologically normal nervous system, and display only defects in MT
stability and organisation in small-calibre axons
(Harada et al., 1994
). The
mild phenotype of tau in mice has been proposed to be due to
functional redundancy with the closely related MAP2, but this cannot be the
case in Drosophila, which does not contain a MAP2 homologue. It may
be redundant with other types of MAP, however, and the best candidate is
Futsch, which has significant structural and functional homology to mammalian
MAP1B (Hummel et al., 2000
;
Roos et al., 2000
). MAP1B
appears to have functional overlap with both Tau and MAP2 in mammals, because
mice that are homozygous for null mutations in map1b and
tau, or map1b and map2, show defects in axonal
elongation, neuronal migration and MT organisation that are much more severe
than in mice lacking only one of these genes
(Takei et al., 2000
;
Teng et al., 2001
).
Another compelling argument that PAR-1 regulates MTs by a different
mechanism from that proposed for the MARKs is that it is required to stabilise
rather than destabilise MTs, at least in epithelial cells. The MTs in follicle
cells are among the most stable in nature, because they are almost completely
resistant to cold or to prolonged colchicine treatments (Gutzeit, 1986;
Theurkauf, 1992) (this study). By contrast, the MTs in par-1 mutant
cells appear to be highly dynamic as: (1) they disappear after brief
colchicine treatments; (2) they depolymerise at 4°C, but re-grow in a few
minutes after return to 25°C; (3) they are lost during fixation, if the
fixative is too weak. Indeed, the instability of the MTs may explain the
discrepancy between our results and those of Cox et al.
(Cox et al., 2001a), as most
MTs in mutant cells disappear during fixation with 4% formaldehyde, but not
with 8% formaldehyde, even though the two fixatives preserve the MTs in
wild-type cells equally well.
The opposite effects of PAR-1 and the MARKs on MT stability may indicate that these closely related kinases have evolved to fulfil distinct functions in invertebrates and mammals. It is also possible, however, that this reflects the different experimental approaches and cell-types that have been used to examine their activities. The MARKs have been assayed by over-expressing them in CHO cells, which are a transformed line of rapidly dividing, undifferentiated and unpolarised cells. By contrast, we have examined the loss-of-function phenotype of PAR-1 in post-mitotic follicle cells, which are highly polarised and differentiated epithelial cells. The two cell types also have very different microtubule cytoskeletons. In CHO cells, microtubules are nucleated from a central centrosome, and are presumably reasonably dynamic, because they disassemble at each mitosis, whereas the follicle cells lose their centrosomes when they form a columnar epithelium, and nucleate a very stable apicobasal array of microtubules. It would therefore be interesting to test the effects on MT stability of disrupting the function of PAR-1 homologues in more similar mammalian cell-types, such as polarised MDCK cells.
In addition to its effect on stability, PAR-1 is required to maintain the
normal organisation of the MTs. The MT arrangement in the follicle cells is
typical of a polarised epithelium, with the minus ends associated with the
apical membrane, and the plus ends at the basal side of the cell
(Gonzalez et al., 1998;
Mogensen, 1999
;
Mogensen et al., 1989
). The
arrangement of minus ends appears to be largely unchanged in par-1
mutant cells, but a marker for the plus ends, Kin:ß-gal, accumulates in
the centre rather than the basal region of the cell. This phenotype is very
similar to that observed in par-1 mutant oocytes, in which the plus
ends become abnormally focussed in the centre of the oocyte, rather than at
the posterior, and there is an increase in the density of MTs around the
cortex (Shulman et al., 2000
).
Thus, PAR-1 may regulate the MTs in the same way in the two cell types, and
most probably acts primarily on the plus ends. PAR-1 may also have some effect
on the distribution of the minus ends of MT in the oocyte, as bicoid
mRNA, which is believed to be transported towards minus ends, is found around
the lateral cortex of mutant oocytes, rather exclusively at the anterior
(Benton et al., 2002
). Although
we cannot rule out the possibility that there is also an effect on the minus
ends in mutant follicle cells, this is not detectable using Nod:ß-gal as
a marker.
It seems paradoxical that the loss of PAR-1 should increase the density of
MTs in follicle cells, while decreasing their stability, but one possible
explanation is suggested by studies in mammalian cells on populations of
stable MTs that are marked by detyrosinated -tubulin
(Bulinski and Gundersen, 1991
;
Webster et al., 1987
). These
MTs are resistant to nocadazole-induced depolymerisation, and fail to
incorporate new tubulin subunits, leading to the proposal that they are capped
at their plus ends in a way that prevents both the addition and loss of
tubulin (Infante et al., 2000
;
Schulze and Kirschner, 1987
;
Webster et al., 1987
). Thus,
it is possible that PAR-1 stabilises the MTs in the follicle cells by capping
plus ends when they reach the basal cortex, thereby preventing them from
either growing or shrinking. If the conditions inside the cell favour MT
polymerisation, the loss of the PAR-1-dependent cap would allow the plus ends
to continue to grow once they reach the basal cortex. This could account for
both the increase in MT density and the redistribution of plus ends to the
centre of mutant cells. However, the uncapped MTs would rapidly shrink under
conditions that favour MT depolymerisation, such as cold or colchicine
treatment, explaining why the MTs disappear in mutant cells.
par-1 null clones also show fully penetrant and cell-autonomous
increases in the recruitment of ß-spectrin and actin to the lateral
cortex. Like the microtubule phenotype, these effects appear to be independent
of the defect in apicobasal membrane polarity, as the latter is much less
penetrant. These phenotypes may therefore reflect a third distinct function of
the kinase. It is also possible, however, that the MT defects are a
consequence of the changes in actin organisation or vice versa. In this
context, it is interesting to note that Rho family GTPases, which are major
regulators of the actin cytoskeleton, have also recently been found to control
the capping of MT plus ends at the leading edge of migrating cells
(Gundersen, 2002). The Rac and
Cdc42 effector, IQGAP, interacts with the plus end binding protein, CLIP170,
to stabilise MTs transiently, whereas the Rho effector, mDia, leads to the
formation of more stable MTs, perhaps through the plus-end binding protein EB1
(Cook et al., 1998
;
Fukata et al., 2002
;
Palazzo et al., 2001
). Given
that PAR-1 does not appear to function through the obvious candidate, Tau, it
would be interesting to test whether this kinase acts through either of these
pathways to regulate MT organisation in epithelial cells.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Harvard Medical School and Brigham and Women's Hospital,
Department of Pathology, 221 Longwood Ave, LMRC 514, Boston, MA 02115, USA
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REFERENCES |
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Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E. H. and Simons, K. (1989). The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J. Cell Biol. 109,2817 -2828.[Abstract]
Bateman, J., Reddy, R. S., Saito, H. and van Vactor, D. (2001). The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium. Curr. Biol. 11,1317 -1327.[CrossRef][Medline]
Baum, B., Li, W. and Perrimon, N. (2000). A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. Curr. Biol. 10,964 -973.[CrossRef][Medline]
Benton, R., Palacios, I. M. and St Johnston, D. (2002). Drosophila 14-3-3/PAR-5 is an essential mediator of PAR-1 function in axis formation. Dev. Cell 3, 659-671.[Medline]
Böhm, H., Brinkmann, V., Drab, M., Henske, A. and Kurzchalia, V. (1997). Mammalian homologues of C. elegans PAR-1 are asymmetrically localized in epithelial cells and may influence their polarity. Curr. Biol. 7, 603-606.[Medline]
Bre, M. H., Pepperkok, R., Hill, A. M., Levilliers, N., Ansorge, W., Stelzer, E. H. and Karsenti, E. (1990). Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells. J. Cell Biol. 111,3013 -3021.[Abstract]
Brower, D. L., Wilcox, M., Piovant, M., Smith, R. J. and Reger, L. A. (1984). Related cell-surface antigens expressed with positional specificity in Drosophila imaginal discs. Proc. Natl. Acad. Sci. USA 81,7485 -7489.[Abstract]
Bulinski, J. C. and Gundersen, G. G. (1991). Stabilization of post-translational modification of microtubules during cellular morphogenesis. BioEssays 13,285 -293.[Medline]
Byers, T. J., Dubreuil, R., Branton, D., Kiehart, D. P. and Goldstein, L. S. (1987). Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins. J. Cell Biol. 105,2103 -2110.[Abstract]
Byers, T. J., Husain-Chishti, A., Dubreuil, R. R., Branton, D. and Goldstein, L. S. (1989). Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin. J. Cell Biol. 109,1633 -1641.[Abstract]
Cha, B., Koppetsch, B. S. and Theurkauf, W. E. (2001). In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway. Cell 106,35 -46.[Medline]
Cha, B. J., Serbus, L. R., Koppetsch, B. S. and Theurkauf, W. E. (2002). Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior. Nat. Cell Biol. 22, 22.
Chapin, S. J. and Bulinski, J. C. (1992). Microtubule stabilization by assembly-promoting microtubule-associated proteins: a repeat performance. Cell Motil. Cytoskel. 23,236 -243.[Medline]
Chou, T.-B., Noll, E. and Perrimon, N. (1993).
Autosomal P[ovoD1] dominant female-sterile insertions in
Drosophila and their use in generating germ-line chimeras.
Development 119,1359
-1369.
Chou, T.-B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics
144,1673
-1679.
Clark, I., Giniger, E., Ruohola-Baker, H., Jan, L. and Jan, Y. (1994). Transient posterior localisation of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[Medline]
Clark, I., Jan, L. Y. and Jan, Y. N. (1997).
Reciprocal localization of Nod and kinesin fusion proteins indicates
microtubule polarity in the Drosophila oocyte, epithelium, neuron and
muscle. Development 124,461
-470.
Cook, T. A., Nagasaki, T. and Gundersen, G. G.
(1998). Rho guanosine triphosphatase mediates the selective
stabilization of microtubules induced by lysophosphatidic acid. J.
Cell Biol. 141,175
-185.
Cox, D. N., Lu, B., Sun, T., Williams, L. T. and Jan, Y. N. (2001a). Drosophila par-1 is required for oocyte differentiation and microtubule organization. Curr. Biol. 11,75 -87.[CrossRef][Medline]
Cox, D. N., Seyfried, S. A., Jan, L. Y. and Jan, Y. N.
(2001b). Bazooka and atypical protein kinase C are required to
regulate oocyte differentiation in the Drosophila ovary.
Proc. Natl. Acad. Sci. USA
98,14475
-14480.
Drewes, G., Ebneth, A., Preuss, U., Mandelkow, E. M. and Mandelkow, E. (1997). MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89,297 -308.[Medline]
Drewes, G., Trinczek, B., Illenberger, S., Biernat, J.,
Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E. M. and Mandelkow, E.
(1995). Microtubule-associated protein/microtubule
affinity-regulating kinase (p110mark). A novel protein kinase that regulates
tau-microtubule interactions and dynamic instability by phosphorylation at the
Alzheimer-specific site serine 262. J. Biol. Chem.
270,7679
-7688.
Ebneth, A., Drewes, G. and Mandelkow, E. (1999). Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells. Cell Motil. Cytoskel. 44,209 -224.[CrossRef][Medline]
Etemad-Moghadam, B., Guo, S. and Kemphues, K. J. (1995). Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 83,743 -752.[Medline]
Fehon, R. G., Johansen, K., Rebay, I. and Artavanis-Tsakonas, S. (1991). Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J. Cell Biol. 113,657 -669.[Abstract]
Fukata, M., Watanabe, T., Noritake, J., Nakagawa, M., Yamaga, M., Kuroda, S., Matsuura, Y., Iwamatsu, A., Perez, F. and Kaibuchi, K. (2002). Rac1 and Cdc42 Capture Microtubules through IQGAP1 and CLIP-170. Cell 109,873 -885.[Medline]
Gonzalez, C., Tavosanis, G. and Mollinari, C.
(1998). Centrosomes and microtubule organisation during
Drosophila development. J. Cell Sci.
111,2697
-2706.
Gundersen, G. G. (2002). Microtubule capture: IQGAP and CLIP-170 expand the repertoire. Curr. Biol. 12,R645 -R647.[CrossRef][Medline]
Guo, S. and Kemphues, K. J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81,611 -620.[Medline]
Harada, A., Oguchi, K., Okabe, S., Kuno, J., Terada, S., Ohshima, T., Sato-Yoshitake, R., Takei, Y., Noda, T. and Hirokawa, N. (1994). Altered microtubule organization in small-calibre axons of mice lacking tau protein. Nature 369,488 -491.[CrossRef][Medline]
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Heidary, G. and Fortini, M. E. (2001). Identification and characterization of the Drosophila tau homolog. Mech. Dev. 108,171 -178.[CrossRef][Medline]
Hortsch, M., Patel, N. H., Bieber, A. J., Traquina, Z. R. and Goodman, C. S. (1990). Drosophila neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis. Development 110,1327 -1340.[Abstract]
Huang, J. and Raff, J. W. (1999). The
disappearance of cyclin B at the end of mitosis is regulated spatially in
Drosophila cells. EMBO J.
18,2184
-2195.
Hummel, T., Krukkert, K., Roos, J., Davis, G. and Klambt, C. (2000). Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron 26,357 -370.[Medline]
Hung, T. J. and Kemphues, K. J. (1999). PAR-6
is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in
Caenorhabditis elegans embryos. Development
126,127
-135.
Huynh, J.-R., Petronczki, M., Knoblich, J. A. and St Johnston, D. (2001a). Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila. Curr. Biol. 11,901 -906.[CrossRef][Medline]
Huynh, J. R., Shulman, J. M., Benton, R. and St Johnston, D.
(2001b). PAR-1 is required for the maintenance of oocyte fate in
Drosophila. Development
128,1201
-1209.
Illenberger, S., Drewes, G., Trinczek, B., Biernat, J., Meyer,
H. E., Olmsted, J. B., Mandelkow, E. M. and Mandelkow, E.
(1996). Phosphorylation of microtubule-associated proteins MAP2
and MAP4 by the protein kinase p110mark. Phosphorylation sites and regulation
of microtubule dynamics. J. Biol. Chem.
271,10834
-10843.
Infante, A. S., Stein, M. S., Zhai, Y., Borisy, G. G. and
Gundersen, G. G. (2000). Detyrosinated (Glu) microtubules are
stabilized by an ATP-sensitive plus-end cap. J. Cell
Sci. 113,3907
-3919.
Izumi, Y., Hirose, T., Tamai, Y., Hirai, S., Nagashima, Y.,
Fujimoto, T., Tabuse, Y., Kemphues, K. J. and Ohno, S.
(1998). An atypical PKC directly associates and colocalizes at
the epithelial tight junction with ASIP, a mammalian homologue of
Caenorhabditis elegans polarity protein PAR-3. J. Cell
Biol. 143,95
-106.
Keller, P. and Simons, K. (1997). Post-Golgi
biosynthetic trafficking. J. Cell Sci.
110,3001
-3009.
Kemphues, K. J., Priess, J. R., Morton, D. G. and Cheng, N. S. (1988). Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52,311 -320.[Medline]
Knust, E. (2000). Control of epithelial cell shape and polarity. Curr. Opin. Genet. Dev. 10,471 -475.[CrossRef][Medline]
Kuchinke, U., Grawe, F. and Knust, E. (1998). Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka. Curr. Biol. 8,1357 -1365.[Medline]
Lee, J. K., Brandin, E., Branton, D. and Goldstein, L. S.
(1997). alpha-Spectrin is required for ovarian follicle monolayer
integrity in Drosophila melanogaster.
Development 124,353
-362.
Lee, V. M., Goedert, M. and Trojanowski, J. Q. (2001). Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24,1121 -1159.[CrossRef][Medline]
Luschnig, S., Krauss, J., Bohmann, K., Desjeux, I. and Nusslein-Volhard, C. (2000). The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases. Mol. Cell 5,231 -241.[Medline]
Martin, S. G. and St Johnston, D. (2003). A role for LKB1 in Drosophila anterior-posterior axis formation and epithelial polarity. Nature 421,379 -384.[CrossRef][Medline]
Matus, A. (1991). Microtubule-associated proteins and neuronal morphogenesis. J. Cell Sci. Suppl. 15,61 -67.[Medline]
Micklem, D. R., Dasgupta, R., Elliott, H., Gergely, F., Davidson, C., Brand, A., González-Reyes, A. and St Johnston, D. (1997). The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7, 468-478.[Medline]
Mogensen, M. M. (1999). Microtubule release and capture in epithelial cells. Biol. Cell 91,331 -341.[CrossRef][Medline]
Mogensen, M. M., Tucker, J. B. and Stebbings, H. (1989). Microtubule polarities indicate that nucleation and capture of microtubules occurs at cell surfaces in Drosophila. J. Cell Biol. 108,1445 -1452.[Abstract]
Mooseker, M. S. (1985). Organization, chemistry, and assembly of the cytoskeletal apparatus of the intestinal brush border. Annu. Rev. Cell Biol. 1, 209-241.[CrossRef][Medline]
Mostov, K. E., Verges, M. and Altschuler, Y. (2000). Membrane traffic in polarized epithelial cells. Curr. Opin. Cell Biol. 12,483 -490.[CrossRef][Medline]
Müller, H.-A. J. (2000). Genetic control of epithelial cell polarity: lessons from Drosophila. Dev. Dyn. 218,52 -67.[CrossRef][Medline]
Muller, H. A. and Wieschaus, E. (1996). armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila. J. Cell Biol. 134,149 -163.[Abstract]
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994). A Drosophila homolog of cadherin associated with armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165,716 -726.[CrossRef][Medline]
Palazzo, A. F., Cook, T. A., Alberts, A. S. and Gundersen, G. G. (2001). mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 3, 723-729.[CrossRef][Medline]
Petronczki, M. and Knoblich, J. A. (2001). DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43-49.[CrossRef][Medline]
Preston, C. R., Sved, J. A. and Engels, W. R.
(1996). Flanking duplications and deletions associated with
P-induced male recombination in Drosophila.
Genetics 144,1623
-1638.
Quaranta, V. (1990). Epithelial integrins. Cell Differ. Dev. 32,361 -365.[CrossRef][Medline]
Queenan, A. M., Ghabrial, A. and Schüpbach, T.
(1997). Ectopic activation of torpedo/Egfr, a
Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and
the embryo. Development
124,3871
-3880.
Riggleman, B., Schedl, P. and Wieschaus, E. (1990). Spatial expression of the Drosophila segment polarity gene armadillo is post-transcriptionally regulated by wingless. Cell 63,549 -560.[Medline]
Roos, J., Hummel, T., Ng, N., Klambt, C. and Davis, G. W. (2000). Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth. Neuron 26,371 -382.[Medline]
Rørth, P. (1998). Gal4 in the Drosophila female germline. Mech. Dev. 78,113 -118.[CrossRef][Medline]
Schuldt, A. J., Adams, J. H. J., Davidson, C. M., Micklem, D.
R., St Johnston, D. and Brand, A. (1998). Miranda mediates
the asymmetric protein and RNA localisation in the developing nervous system.
Genes Dev. 12,1847
-1857.
Schulze, E. and Kirschner, M. (1987). Dynamic and stable populations of microtubules in cells. J. Cell Biol. 104,277 -288.[Abstract]
Shulman, J. M., Benton, R. and St. Johnston, D. (2000). The Drosophila homolog of C. elegans PAR-1 organizes the oocyte cytoskeleton and directs oskar mRNA localisation to the posterior pole. Cell 101, 1-20.[Medline]
Sun, T.-Q., Lu, B., Feng, J.-J., Reinhard, C., Jan, Y. N., Fantl, W. J. and Williams, L. T. (2001). PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling. Nat. Cell Biol. 3,628 -636.[CrossRef][Medline]
Suzuki, A., Yamanaka, T., Hirose, T., Manabe, N., Mizuno, K.,
Shimizu, M., Akimoto, K., Izumi, Y., Ohnishi, T. and Ohno, S.
(2001). Atypical protein kinase C is involved in the
evolutionarily conserved PAR protein complex and plays a critical role in
establishing epithelia-specific junctional structures. J. Cell
Biol. 152,1183
-1196.
Tabuse, Y., Izumi, Y., Piano, F., Kemphues, K. J., Miwa, J. and
Ohno, S. (1998). Atypical protein kinase C cooperates with
PAR-3 to establish embryonic polarity in Caenorhabditis elegans.
Development 125,3607
-3614.
Takei, Y., Teng, J., Harada, A. and Hirokawa, N.
(2000). Defects in axonal elongation and neuronal migration in
mice with disrupted tau and map1b genes. J. Cell Biol.
150,989
-1000.
Tanentzapf, G., Smith, C., McGlade, J. and Tepass, U.
(2000). Apical, lateral, and basal polarization cues contribute
to the development of the follicular epithelium during Drosophila
oogenesis. J. Cell Biol.
151,891
-904.
Teng, J., Takei, Y., Harada, A., Nakata, T., Chen, J. and
Hirokawa, N. (2001). Synergistic effects of MAP2 and MAP1B
knockout in neuronal migration, dendritic outgrowth, and microtubule
organization. J. Cell Biol.
155, 65-76.
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35,747 -784.[CrossRef][Medline]
Tepass, U., Theres, C. and Knust, E. (1990). crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61,787 -799.[Medline]
Theurkauf, W. E. (1994). Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. Methods Cell Biol. 44,489 -505.[Medline]
Theurkauf, W. E., Smiley, S., Wong, M. L. and Alberts, B. M.
(1992). Reorganization of the cytoskeleton during
Drosophila oogenesis: implications for axis specification and
intercellular transport. Development
115,923
-936.
Thomas, G. H. and Kiehart, D. P. (1994). Beta
heavy-spectrin has a restricted tissue and subcellular distribution during
Drosophila embryogenesis. Development
120,2039
-2050.
Tomancak, P., Piano, F., Riechmann, V., Gunsalus, K. C., Kemphues, K. J. and Ephrussi, A. (2000). A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation. Nat. Cell Biol. 2,458 -460.[CrossRef][Medline]
Watts, J. L., Etemad-Moghadam, B., Guo, S., Boyd, L., Draper, B.
W., Mello, C. C., Priess, J. R. and Kemphues, K. J. (1996).
par-6, a gene involved in the establishment of asymmetry in early
C. elegans embryos, mediates the asymmetric localization of PAR-3.
Development 122,3133
-3140.
Webster, D. R., Gundersen, G. G., Bulinski, J. C. and Borisy, G. G. (1987). Differential turnover of tyrosinated and detyrosinated microtubules. Proc. Natl. Acad. Sci. USA 84,9040 -9044.[Abstract]
Wodarz, A., Ramrath, A., Grimm, A. and Knust, E.
(2000). Drosophila atypical protein kinase C associates
with Bazooka and controls polarity of epithelia and neuroblasts. J.
Cell Biol. 150,1361
-1374.
Xu, T. and Rubin, G. (1993). Analysis of
genetic mosaics in developing an adult Drosphila tissues.
Development 117,1223
-1237.
Yeaman, C., Grindstaff, K. K. and Nelson, W. J.
(1999). New perspectives on mechanisms involved in generating
epithelial cell polarity. Physiol. Rev.
79, 73-98.